Single cell secretome analysis

ABSTRACT

Systems, methods, compositions, and kits for measuring secreted factors from cells are disclosed herein, including those capable of determining single cell secretion activity and protein expression and/or gene expression simultaneously. Disclosed herein include solid supports comprising a plurality of capture probes capable of specifically binding to at least one of the plurality of secreted factors secreted by a single cell. Also disclosed herein include secreted factor-binding reagents capable of specifically binding to a secreted factor bound by a capture probe. A secreted factor-binding reagent can comprise a secreted factor-binding reagent specific oligonucleotide comprising a unique factor identifier sequence for the secreted factor-binding reagent.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 63/125,629, filed Dec. 15, 2020,the content of this related application is incorporated herein byreference in its entirety for all purposes.

BACKGROUND Field

The present disclosure relates generally to the field of molecularbiology, for example determining the secreted molecule profiles of cellsusing molecular barcoding.

Description of the Related Art

Current technology allows measurement of gene expression of single cellsin a massively parallel manner (e.g., >10000 cells) by attaching cellspecific oligonucleotide barcodes to poly(A) mRNA molecules fromindividual cells as each of the cells is co-localized with a barcodedreagent bead in a compartment. Gene expression may affect proteinexpression and the secretion of molecules. Protein-protein interactionmay affect gene expression and protein expression as well as secretionof molecules by cells. Cytokines and other molecules released by thecell are of keen interest to immunologists and other cell biologists.Traditional methods for detecting and measuring secreted proteins aretypically measured in bulk (rather than at the single cell level). Forexample, currently available methods include bead-based assays and ELISAfor studying secreted factors in bulk. Therefore, single cellquantification and cellular phenotype analysis are missing in the data.As with the comparison of flow cytometry to traditional western blots,there is tremendous value in studying the individual cells from aheterogenous mixture of cells. There is an increasing need to correlatespecific secretion activity with complex cell phenotype. Currentlyavailable methods for detecting secreted proteins have a limitation onthe number of proteins that can be detected due to the number offluorescence markers that can used in the microscope or flow cytometryanalysis. Moreover, such methods are less quantitative than desired dueto limitations in measuring fluorescence intensity differences. There isa need for systems and methods that can quantitatively analyze thenumber of copies of a secreted factor secreted by a single cell. Thereis a need for systems and methods that can quantitatively analyze thenumber of copies of a secreted factor secreted by a single cell andsimultaneously measure protein expression and/or gene expression.

SUMMARY

Disclosed herein include methods of measuring the number of copies of asecreted factor secreted by a single cell. The method can comprise:contacting one or more single cells with a first plurality of firstsolid supports, the one or more single cells are capable of secreting aplurality of secreted factors, each first solid support comprises aplurality of capture probes capable of specifically binding to at leastone of the plurality of secreted factors secreted by a single cell. Themethod can comprise: contacting the first solid support with a pluralityof secreted factor-binding reagents each capable of specifically bindingto a secreted factor bound by a capture probe, each of the plurality ofsecreted factor-binding reagents comprises a secreted factor-bindingreagent specific oligonucleotide comprising a unique factor identifiersequence for the secreted factor-binding reagent. The method cancomprise: contacting a plurality of oligonucleotide barcodes with thesecreted factor-binding reagent specific oligonucleotides forhybridization, the oligonucleotide barcodes each comprise a firstmolecular label. The method can comprise: extending the plurality ofoligonucleotide barcodes hybridized to the secreted factor-bindingreagent specific oligonucleotides to generate a plurality of barcodedsecreted factor-binding reagent specific oligonucleotides eachcomprising a sequence complementary to at least a portion of the uniquefactor identifier sequence and the first molecular label. The method cancomprise: obtaining sequence information of the plurality of barcodedsecreted factor-binding reagent specific oligonucleotides, or productsthereof, to determine the number of copies of the at least one secretedfactor secreted by each of the one or more single cells. In someembodiments, the one or more single cells comprises T cells, B cells,tumor cells, myeloid cells, blood cells, normal cells, fetal cells,maternal cells, or a mixture thereof.

In some embodiments, contacting one or more single cells with a firstplurality of first solid supports comprises: partitioning the one ormore single cells and the first plurality of first solid supports to aplurality of first partitions, a first partition of the plurality offirst partitions comprises a single cell of the one or more single cellsand a single first solid support of the first plurality of first solidsupports. In some embodiments, the method comprises, prior to contactingthe first solid support with a plurality of secreted factor-bindingreagents: pooling the single first solid supports from each firstpartition of the plurality of first partitions to generate a secondplurality of first solid supports. In some embodiments, contacting thefirst solid support with a plurality of secreted factor-binding reagentscomprises contacting the second plurality of first solid supports withthe plurality of secreted factor-binding reagents. In some embodiments,the method comprises, after contacting the second plurality of firstsolid supports with the plurality of secreted factor-binding reagents,removing one or more secreted factor-binding reagents of the pluralityof secreted factor-binding reagents that are not contacted with thesecond plurality of first solid supports to generate a third pluralityof first solid supports. In some embodiments, removing the one or moresecreted factor-binding reagents not contacted with the second pluralityof first solid supports comprises: removing the one or more secretedfactor-binding reagents not contacted with the respective at least oneof the secreted factor bound by a capture probe. In some embodiments,contacting a plurality of oligonucleotide barcodes with the secretedfactor-binding reagent specific oligonucleotides for hybridizationcomprises: partitioning the third plurality of first solid supports to aplurality of second partitions, a second partition of the plurality ofsecond partitions comprises a single first solid support from the thirdplurality of first solid supports; and in the second partitioncomprising the single first solid support, contacting a plurality ofoligonucleotide barcodes with the secreted factor-binding reagentspecific oligonucleotides for hybridization.

Disclosed herein include methods of measuring the number of copies of asecreted factor secreted by a single cell and the number of copies of anucleic acid target in a single cell. The method can comprise:contacting one or more single cells with a first plurality of secondsolid supports to form one or more single cells associated with a secondsolid support, the one or more single cells comprise a surface cellulartarget and copies of a nucleic acid target, the one or more single cellsare capable of secreting a plurality of secreted factors, each secondsolid support comprises a plurality of capture probes and a plurality ofanchor probes, each of the plurality of anchor probes is capable ofspecifically binding to the surface cellular target, and the captureprobe is capable of specifically binding to at least one of theplurality of secreted factors secreted by a single cell. The method cancomprise: contacting the one or more single cells associated with asecond solid support with a plurality of secreted factor-bindingreagents capable of specifically binding to a secreted factor bound by acapture probe, each of the plurality of secreted factor-binding reagentscomprises a secreted factor-binding reagent specific oligonucleotidecomprising a unique factor identifier sequence for the secretedfactor-binding reagent. The method can comprise: contacting a pluralityof oligonucleotide barcodes with the secreted factor-binding reagentspecific oligonucleotides and the copies of the nucleic acid target forhybridization, the oligonucleotide barcodes each comprise a firstmolecular label. The method can comprise: extending the plurality ofoligonucleotide barcodes hybridized to the copies of a nucleic acidtarget to generate a plurality of barcoded nucleic acid molecules eachcomprising a sequence complementary to at least a portion of the nucleicacid target and the first molecular label. The method can comprise:extending the plurality of oligonucleotide barcodes hybridized to thesecreted factor-binding reagent specific oligonucleotides to generate aplurality of barcoded secreted factor-binding reagent specificoligonucleotides each comprising a sequence complementary to at least aportion of the unique factor identifier sequence and the first molecularlabel. The method can comprise: obtaining sequence information of theplurality of barcoded nucleic acid molecules, or products thereof, todetermine the copy number of the nucleic acid target in each of the oneor more single cells. The method can comprise: obtaining sequenceinformation of the plurality of barcoded secreted factor-binding reagentspecific oligonucleotides, or products thereof, to determine the numberof copies of the at least one secreted factor secreted by each of theone or more single cells (e.g., T cells, B cells, tumor cells, myeloidcells, blood cells, normal cells, fetal cells, maternal cells, or amixture thereof).

In some embodiments, contacting one or more single cells with a firstplurality of second solid supports to form one or more single cellsassociated with a second solid support comprises: partitioning the oneor more single cells and the plurality of second solid supports to aplurality of first partitions, a first partition of the plurality offirst partitions comprises a single cell of the one or more single cellsand a single second solid support of the plurality of second solidsupports, the single cell is capable of becoming associated with asecond solid support via the anchor probe binding to the surfacecellular target. In some embodiments, the method comprises, prior tocontacting the one or more single cells associated with a second solidsupport with a plurality of secreted factor-binding reagents: poolingthe single cells associated with a second solid support from each firstpartition of the plurality of first partitions to generate a firstplurality of single cells associated with a second solid support.

In some embodiments, contacting the one or more single cells associatedwith a second solid support with a plurality of secreted factor-bindingreagents comprises contacting the first plurality of single cellsassociated with a second solid support with the plurality of secretedfactor-binding reagents. In some embodiments, the method comprises,after contacting the first plurality of single cells associated with asecond solid support with the plurality of secreted factor-bindingreagents, removing one or more secreted factor-binding reagents of theplurality of secreted factor-binding reagents that are not contactedwith the first plurality of single cells associated with a second solidsupport to generate a second plurality of single cells associated with asecond solid support. In some embodiments, removing the one or moresecreted factor-binding reagents not contacted with the first pluralityof single cells associated with a second solid support comprises:removing the one or more secreted factor-binding reagents not contactedwith the respective at least one of the secreted factor bound by acapture probe.

The method can comprise: prior to contacting a plurality ofoligonucleotide barcodes with the secreted factor-binding reagentspecific oligonucleotides and the copies of the nucleic acid target forhybridization: partitioning the second plurality of single cellsassociated with a second solid support to a plurality of secondpartitions, a second partition of the plurality of second partitionscomprises a single cell and a single second solid support from thesecond plurality of single cells associated with a second solid support;in the second partition comprising the single cell and the single secondsolid support, contacting a plurality of oligonucleotide barcodes withthe secreted factor-binding reagent specific oligonucleotides and thecopies of the nucleic acid target for hybridization. In someembodiments, the method comprises lysing the single cell in the secondpartition. Lysing the single cell can comprise heating the sample,contacting the sample with a detergent, changing the pH of the sample,or any combination thereof.

The at least one secreted factor can comprise a lymphokine, aninterleukin, a chemokine, or any combination thereof. For example, thesecreted factor can be a cytokine, a hormone, a molecular toxin, or anycombination thereof. In some embodiments, the at least one secretedfactor comprises a nerve growth factor, a hepatic growth factor, afibroblast growth factor, a vascular endothelial growth factor, aplatelet-derived growth factor, a transforming growth factor, anosteoinductive factor, an interferon, a colony stimulating factor, orany combination thereof. The at least one secreted factor can compriseangiogenin, angiopoietin-1, angiopoietin-2, bNGF, cathepsin S,Galectin-7, GCP-2, G-CSF, GM-CSF, PAI-1, PDGF-AA, PDGF-BB, PDGF-AB,PlGF, PlGF-2, SDF-1, Tie2, VEGF-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2,VEGF-R3, 6Ckine, angiopoietin-1, angiopoietin-2, BLC, BRAK, CD186,ENA-78, Eotaxin-1, Eotaxin-2, Eotaxin-3, EpCAM, GDF-15, GM-CSF, GRO,HCC-4, I-309, IFN-γ, IL-1α, IL-1β, IL-1R4 (ST2), IL-2, IL-2R, IL-3,IL-3Rα, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-8 RB, IL-11, IL-12, IL-12p40,IL-12p70, IL-13, IL-13 R1, IL-13R2, IL-15, IL-15Rα, IL-16, IL-17,IL-17C, IL-17E, IL-17F, IL-17R, IL-18, IL-18BPa, IL-18 Rα, IL-20, IL-23,IL-27, IL-28, IL-31, IL-33, IP-10, I-TAC, LIF, LIX, LRP6, MadCAM-1,MCP-1, MCP-2, MCP-3, MCP-4, M-CSF, MIF, MIG, MIP-1 gamma, MIP-1α,MIP-1β, MIP-1δ, MIP-3α, MIP-3β, MPIF-1, PARC, PF4, RANTES, Resistin,SCF, SCYB16, TACI, TARC, TSLP, TNF-α, TNF-R1, TRAIL-R4, TREM-1, ActivinA, Amphiregulin, Axl, BDNF, BMP4, cathepsin S, EGF, FGF-1, FGF-2, FGF-7,FGF-21, Follistatin, Galectin-7, Gas6, GDF-15, HB-EGF, HGF, IGFBP-1,IGFBP-3, LAP, NGF R, NrCAM, NT-3, NT-4, PAI-1, TGF-α, TGF-β, TGF-β3,TRAIL-R4, ADAMTS1, cathepsin S, FGF-2, Follistatin, Galectin-7, GCP-2,GDF-15, IGFBP-6, LIF, MMP-9, pro-MMP9, RANK, RANKL, RANTES, SDF-1,CXCR4, or any combination thereof.

In some embodiments, the secreted factor-binding reagent and the captureprobe are capable of binding to distinct epitopes of the same secretedfactor. In some embodiments, one or more of the secreted factor-bindingreagents, the capture probe, and the anchor probe comprise an antibodyor fragment thereof. In some embodiments, the antibody or fragmentthereof comprises a monoclonal antibody. In some embodiments, theantibody or fragment thereof comprises a Fab, a Fab′, a F(ab′)₂, a Fv, ascFv, a dsFv, a diabody, a triabody, a tetrabody, a multispecificantibody formed from antibody fragments, a single-domain antibody(sdAb), a single chain comprising complementary scFvs (tandem scFvs) orbispecific tandem scFvs, an Fv construct, a disulfide-linked Fv, a dualvariable domain immunoglobulin (DVD-Ig) binding protein or a nanobody,an aptamer, an affibody, an affilin, an affitin, an affimer, analphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domainpeptide, a monobody, or any combination thereof. In some embodiments,the capture probe and/or the anchor probe is conjugated to the firstsolid support and/or the second solid support by a 1,3-dipolarcycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilicsubstitution reaction, a non-aldol type carbonyl reaction, an additionto carbon-carbon multiple bond, an oxidation reaction, a click reaction,or any combination thereof.

The surface cellular target can comprise a carbohydrate, a lipid, aprotein, an extracellular protein, a cell-surface protein, a cellmarker, a B-cell receptor, a T-cell receptor, a major histocompatibilitycomplex, a tumor antigen, a receptor, an intracellular protein, or anycombination thereof. For example, the surface cellular target cancomprise a carbohydrate, a lipid, a protein, or any combination thereof.In some embodiments, the surface cellular target comprises CD1a, CD1b,CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a,CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15,CD15u, CD15s, CD15su, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22,CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34,CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d,CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48,CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54,CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L,CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e,CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77,CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85a, CD85d, CD85j, CD85k,CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97,CD98, CD99, CD99R, CD100, CD101, CD102,CD103, CD104, CD105, CD106,CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115,CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122,CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131, CD132, CD133,CD134, CD135, CD136, CD137, CD138, CD139, CD140a, CD140b, CD141, CD142,CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152,CD153, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158e, CD158i,CD158k, CD159a, CD159c, CD160, CD161, CD162, CD163, CD164, CD165, CD166,CD167a, CD167b, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g,CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180,CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194,CD195, CD196, CD197, CDw198, CD199, CD200, CD201, CD202b, CD203c, CD204,CD205, CD206, CD207, CD208, CD209, CD210, CDw210b, CD212, CD213a1,CD213a2, CD215, CD217a, CD218a, CD218b, CD220, CD221, CD222, CD223,CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233,CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240CE, CD240DCE,CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249,CD252, CD253, CD254, CD256, CD266, CD267, CD268, CD269, CD270, CD271,CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281,CD282, CD283, CD284, CD286, CD289, CD290, CD292, CDw293, CD294, CD295,CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303,CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308,CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321,CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333,CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350,CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362,CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, BCMA, aHLA protein, β2-microglobulin, or any combination thereof.

In some embodiments, the plurality of oligonucleotide barcodes areassociated with a third solid support, and a second partition of theplurality of second partitions comprises a single third solid support.In some embodiments, the first partition and/or second partition is awell or a droplet. In some embodiments, each oligonucleotide barcodecomprises a first universal sequence. In some embodiments, theoligonucleotide barcode comprises a target-binding region comprising acapture sequence. In some embodiments, the target-binding regioncomprises a poly(dT) region. In some embodiments, the secretedfactor-binding reagent specific oligonucleotide comprises a sequencecomplementary to the capture sequence configured to capture the secretedfactor-binding reagent specific oligonucleotide. In some embodiments,the sequence complementary to the capture sequence comprises a poly(dA)region. In some embodiments, the plurality of barcoded secretedfactor-binding reagent specific oligonucleotides comprise a complementof the first universal sequence. In some embodiments, the secretedfactor-binding reagent specific oligonucleotide comprises a seconduniversal sequence.

In some embodiments, obtaining sequence information of the plurality ofbarcoded secreted factor-binding reagent specific oligonucleotides, orproducts thereof, comprises: amplifying the plurality of barcodedsecreted factor-binding reagent specific oligonucleotides, or productsthereof, using a primer capable of hybridizing to the first universalsequence, or a complement thereof, and a primer capable of hybridizingto the second universal sequence, or a complement thereof, to generate aplurality of amplified barcoded secreted factor-binding reagent specificoligonucleotides; and obtaining sequencing data of the plurality ofamplified barcoded secreted factor-binding reagent specificoligonucleotides, or products thereof.

The secreted factor-binding reagent specific oligonucleotide cancomprise a second molecular label. In some embodiments, at least ten ofthe plurality of secreted factor-binding reagent specificoligonucleotides comprise different second molecular label sequences. Insome embodiments, the second molecular label sequences of at least twosecreted factor-binding reagent specific oligonucleotides are different,and the unique identifier sequences of the at least two secretedfactor-binding reagent specific oligonucleotides are identical. In someembodiments, the second molecular label sequences of at least twosecreted factor-binding reagent specific oligonucleotides are different,and the unique identifier sequences of the at least two secretedfactor-binding reagent specific oligonucleotides are different.

In some embodiments, the number of unique first molecular labelsequences associated with the unique factor identifier sequence for thesecreted factor-binding reagent capable of specifically binding to theat least one secreted factor in the sequencing data indicates the numberof copies of the at least one secreted factor secreted by each of theone or more single cells. In some embodiments, the number of uniquesecond molecular label sequences associated with the unique factoridentifier sequence for the secreted factor-binding reagent capable ofspecifically binding to the at least one secreted factor in thesequencing data indicates the number of copies of the at least onesecreted factor secreted by each of the one or more single cells. Insome embodiments, the method comprises determining the number of copiesof the at least one secreted factor secreted by each of the one or moresingle cells based on the number of first molecular labels and/or secondmolecular labels with distinct sequences associated with the pluralityof barcoded secreted factor-binding reagent specific oligonucleotides,or products thereof.

In some embodiments, the method comprises determining the number ofcopies of the at least one secreted factor secreted by each of the oneor more single cells based on the number of first molecular labelsand/or second molecular labels with distinct sequences associated withthe plurality of amplified barcoded secreted factor-binding reagentspecific oligonucleotides, or products thereof. In some embodiments,obtaining the sequence information comprises attaching sequencingadaptors to the plurality of barcoded secreted factor-binding reagentspecific oligonucleotides, or products thereof.

In some embodiments, the secreted factor-binding reagent specificoligonucleotide comprises an alignment sequence adjacent to the poly(dA)region. The alignment sequence can be, one or more nucleotides inlength, or two or more nucleotides in length. For example, the alignmentsequence can (a) comprises a guanine, a cytosine, a thymine, a uracil,or a combination thereof; (b) comprises a poly(dT) sequence, a poly(dG)sequence, a poly(dC) sequence, a poly(dU) sequence, or a combinationthereof; and/or (c) is 5′ to the poly(dA) region.

In some embodiments, the secreted factor-binding reagent specificoligonucleotide is associated with the secreted factor-binding reagentthrough a linker. In some embodiments, the linker comprises a carbonchain. The carbon chain can comprise 2-30 carbons (e.g., 12 carbons). Insome embodiments, the linker comprises 5′ amino modifier C12 (5AmMC12),or a derivative thereof. In some embodiments, the secretedfactor-binding reagent specific oligonucleotide is configured to bedetachable from the secreted factor-binding reagent. For example, themethod can comprise dissociating the secreted factor-binding reagentspecific oligonucleotide from the secreted factor-binding reagent.

In some embodiments, determining the copy number of the nucleic acidtarget in each of the one or more single cells comprises determining thecopy number of the nucleic acid target in each of the one or more singlecells based on the number of first molecular labels with distinctsequences, complements thereof, or a combination thereof, associatedwith the plurality of barcoded nucleic acid molecules, or productsthereof.

In some embodiments, the method comprises: contacting random primerswith the plurality of barcoded nucleic acid molecules, each of therandom primers comprises a third universal sequence, or a complementthereof; and extending the random primers hybridized to the plurality ofbarcoded nucleic acid molecules to generate a plurality of extensionproducts. In some embodiments, the method comprises amplifying theplurality of extension products using primers capable of hybridizing tothe first universal sequence or complements thereof, and primers capableof hybridizing the third universal sequence or complements thereof,thereby generating a first plurality of barcoded amplicons. In someembodiments, amplifying the plurality of extension products comprisesadding sequences of binding sites of sequencing primers and/orsequencing adaptors, complementary sequences thereof, and/or portionsthereof, to the plurality of extension products. In some embodiments,the method comprises determining the copy number of the nucleic acidtarget in each of the one or more single cells based on the number offirst molecular labels with distinct sequences associated with the firstplurality of barcoded amplicons, or products thereof.

In some embodiments, determining the copy number of the nucleic acidtarget in each of the one or more single cells comprises determining thenumber of each of the plurality of nucleic acid targets in each of theone or more single cells based on the number of the first molecularlabels with distinct sequences associated with barcoded amplicons of thefirst plurality of barcoded amplicons comprising a sequence of the eachof the plurality of nucleic acid targets. In some embodiments, thesequence of the each of the plurality of nucleic acid targets comprisesa subsequence of the each of the plurality of nucleic acid targets. Insome embodiments, the sequence of the nucleic acid target in the firstplurality of barcoded amplicons comprises a subsequence of the nucleicacid target.

In some embodiments, the method comprises amplifying the first pluralityof barcoded amplicons using primers capable of hybridizing to the firstuniversal sequence or complements thereof, and primers capable ofhybridizing the third universal sequence or complements thereof, therebygenerating a second plurality of barcoded amplicons. In someembodiments, amplifying the first plurality of barcoded ampliconscomprises adding sequences of binding sites of sequencing primers and/orsequencing adaptors, complementary sequences thereof, and/or portionsthereof, to the first plurality of barcoded amplicons. In someembodiments, the method comprises determining the copy number of thenucleic acid target in each of the one or more single cells based on thenumber of first molecular labels with distinct sequences associated withthe second plurality of barcoded amplicons, or products thereof. In someembodiments, the first plurality of barcoded amplicons and/or the secondplurality of barcoded amplicons comprise whole transcriptomeamplification (WTA) products.

In some embodiments, the method comprises synthesizing a third pluralityof barcoded amplicons using the plurality of barcoded nucleic acidmolecules as templates to generate a third plurality of barcodedamplicons. In some embodiments, synthesizing a third plurality ofbarcoded amplicons comprises performing (1) PCR amplification of theplurality of the barcoded nucleic acid molecules; (2) PCR amplificationusing primers capable of hybridizing to the first universal sequence, ora complement thereof, and a target-specific primer; or both. In someembodiments, the method comprises obtaining sequence information of thethird plurality of barcoded amplicons, or products thereof. Obtainingthe sequence information can comprise attaching sequencing adaptors tothe third plurality of barcoded amplicons, or products thereof. Themethod can comprise determining the copy number of the nucleic acidtarget in each of the one or more single cells based on the number offirst molecular labels with distinct sequences associated with the thirdplurality of barcoded amplicons, or products thereof.

In some embodiments, the nucleic acid target comprises a nucleic acidmolecule. In some embodiments, the nucleic acid molecule comprisesribonucleic acid (RNA), messenger RNA (mRNA), microRNA, smallinterfering RNA (siRNA), RNA degradation product, RNA comprising apoly(A) tail, a sample indexing oligonucleotide, a cellularcomponent-binding reagent specific oligonucleotide, or any combinationthereof. In some embodiments, extending the plurality of oligonucleotidebarcodes comprising extending the plurality of oligonucleotide barcodesusing a reverse transcriptase and/or a DNA polymerase lacking at leastone of 5′ to 3′ exonuclease activity and 3′ to 5′ exonuclease activity.In some embodiments, the DNA polymerase comprises a Klenow Fragment. Insome embodiments, the reverse transcriptase comprises a viral reversetranscriptase (e.g., a murine leukemia virus (MLV) reverse transcriptaseor a Moloney murine leukemia virus (MMLV) reverse transcriptase).

In some embodiments, the first universal sequence, the second universalsequence, and/or the third universal sequence are the same. In someembodiments, the first universal sequence, the second universalsequence, and/or the third universal sequence are different. In someembodiments, the first universal sequence, the second universalsequence, and/or the third universal sequence comprise the binding sitesof sequencing primers and/or a sequencing adaptor, complementarysequences thereof, and/or portions thereof. In some embodiments, thesequencing adaptors comprise a P5 sequence, a P7 sequence, complementarysequences thereof, and/or portions thereof. In some embodiments, thesequencing primers comprise a Read 1 sequencing primer, a Read 2sequencing primer, complementary sequences thereof, and/or portionsthereof. In some embodiments, at least 10 of the plurality ofoligonucleotide barcodes comprise different first molecular labelsequences. In some embodiments, the plurality of oligonucleotidebarcodes each comprise a cell label. In some embodiments, each celllabel of the plurality of oligonucleotide barcodes comprises at least 6nucleotides. In some embodiments, oligonucleotide barcodes associatedwith the same third solid support comprise the same cell label. In someembodiments, oligonucleotide barcodes associated with different thirdsolid supports comprise different cell labels.

In some embodiments, the first solid support, second solid support,and/or third solid support comprises a synthetic particle or a planarsurface. In some embodiments, at least one of the plurality ofoligonucleotide barcodes is immobilized or partially immobilized on thesynthetic particle, or the at least one of the plurality ofoligonucleotide barcodes is enclosed or partially enclosed in thesynthetic particle. The synthetic particle can be disruptable. Thesynthetic particle can comprise a bead. The bead can comprise: asepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, aconjugated bead, a protein A conjugated bead, a protein G conjugatedbead, a protein A/G conjugated bead, a protein L conjugated bead, anoligo(dT) conjugated bead, a silica bead, a silica-like bead, ananti-biotin microbead, an anti-fluorochrome microbead, or anycombination thereof; a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone,and any combination thereof; or a disruptable hydrogel particle.

In some embodiments, each of the plurality of oligonucleotide barcodescomprises a linker functional group, the synthetic particle comprises asolid support functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

In some embodiments, each of the plurality of anchor probes comprises alinker functional group, the synthetic particle comprises a solidsupport functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

In some embodiments, each of the plurality of capture probes comprises alinker functional group, the synthetic particle comprises a solidsupport functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

In some embodiments, the first solid support and/or the second solidsupport is sized and shaped to approximate a cell. In some embodiments,the first solid support and/or the second solid support has thedimensions of a cell. In some embodiments, the cell is a mammalian cell,a yeast cell, an insect cell, a plant cell, a bacterial cell, or anycombination thereof.

Disclosed herein include compositions (e.g., kits). The composition cancomprise: a first solid support comprising a plurality of capture probescapable of specifically binding to at least one of a plurality ofsecreted factors secreted by a single cell; and a plurality of secretedfactor-binding reagents each capable of specifically binding to asecreted factor bound by a capture probe, each of the plurality ofsecreted factor-binding reagents comprises a secreted factor-bindingreagent specific oligonucleotide comprising a unique factor identifiersequence for the secreted factor-binding reagent.

Disclosed herein include compositions (e.g., kits). The composition cancomprise: a second solid support comprising a plurality of captureprobes and a plurality of anchor probes, each of the plurality of anchorprobes is capable of specifically binding to a surface cellular target,and the capture probe is capable of specifically binding to at least oneof a plurality of secreted factors secreted by a single cell; and aplurality of secreted factor-binding reagents each capable ofspecifically binding to a secreted factor bound by a capture probe, eachof the plurality of secreted factor-binding reagents comprises asecreted factor-binding reagent specific oligonucleotide comprising aunique factor identifier sequence for the secreted factor-bindingreagent.

In some embodiments, the secreted factor-binding reagent specificoligonucleotide comprises a second molecular label sequence (e.g., 2-20nucleotides in length). In some embodiments, the second molecular labelsequences of at least two secreted factor-binding reagent specificoligonucleotides are different, and the unique identifier sequences ofthe at least two secreted factor-binding reagent specificoligonucleotides are identical. In some embodiments, the secondmolecular label sequences of at least two secreted factor-bindingreagent specific oligonucleotides are different, and the uniqueidentifier sequences of the at least two secreted factor-binding reagentspecific oligonucleotides are different.

In some embodiments, the secreted factor-binding reagent specificoligonucleotide comprises a second universal sequence. In someembodiments, the second universal sequence comprises a binding site of asequencing primers and/or a sequencing adaptor, complementary sequencesthereof, and/or portions thereof. In some embodiments, the sequencingadaptor comprises a P5 sequence, a P7 sequence, complementary sequencesthereof, and/or portions thereof. In some embodiments, the sequencingprimer comprises a Read 1 sequencing primer, a Read 2 sequencing primer,complementary sequences thereof, and/or portions thereof.

In some embodiments, the secreted factor-binding reagent specificoligonucleotide comprises a poly(dA) region. In some embodiments, thesecreted factor-binding reagent specific oligonucleotide comprises analignment sequence adjacent to the poly(dA) region. The alignmentsequence can be one or more nucleotides in length, or two or morenucleotides in length. The alignment sequence can (a) comprises aguanine, a cytosine, a thymine, a uracil, or a combination thereof; (b)comprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence,a poly(dU) sequence, or a combination thereof; and/or (c) is 5′ to thepoly(dA) region.

In some embodiments, the secreted factor-binding reagent specificoligonucleotide is associated with the secreted factor-binding reagentthrough a linker. In some embodiments, the linker comprises a carbonchain. The carbon chain can comprise 2-30 carbons (e.g., 12 carbons). Insome embodiments, the linker comprises 5′ amino modifier C12 (5AmMC12),or a derivative thereof. In some embodiments, the secretedfactor-binding reagent specific oligonucleotide is attached to thesecreted factor-binding reagent. In some embodiments, the secretedfactor-binding reagent specific oligonucleotide is covalently attachedto the secreted factor-binding reagent. In some embodiments, thesecreted factor-binding reagent specific oligonucleotide isnon-covalently attached to the secreted factor-binding reagent. In someembodiments, the secreted factor-binding reagent specificoligonucleotide is conjugated to the secreted factor-binding reagent. Insome embodiments, the secreted factor-binding reagent specificoligonucleotide isconjugated to the secreted factor-binding reagentthrough a chemical group, such as a UV photocleavable group, astreptavidin, a biotin, an amine, or a combination thereof.

The secreted factor can comprise a lymphokine, an interleukin, achemokine, or any combination thereof. For example, the secreted factorcan comprise a cytokine, a hormone, a molecular toxin, or anycombination thereof. In some embodiments, the secreted factor comprisesa nerve growth factor, a hepatic growth factor, a fibroblast growthfactor, a vascular endothelial growth factor, a platelet-derived growthfactor, a transforming growth factor, an osteoinductive factor, aninterferon, a colony stimulating factor, or any combination thereof.

In some embodiments, the secreted factor-binding reagents and thecapture probe are capable of binding to distinct epitopes of the samesecreted factor. In some embodiments, one or more of the secretedfactor-binding reagents, the capture probe, and the anchor probecomprise an antibody or fragment thereof. In some embodiments, theantibody or fragment thereof comprises a monoclonal antibody. In someembodiments, the antibody or fragment thereof comprises a Fab, a Fab′, aF(ab′)₂, a Fv, a scFv, a dsFv, a diabody, a triabody, a tetrabody, amultispecific antibody formed from antibody fragments, a single-domainantibody (sdAb), a single chain comprising complementary scFvs (tandemscFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linkedFv, a dual variable domain immunoglobulin (DVD-Ig) binding protein or ananobody, an aptamer, an affibody, an affilin, an affitin, an affimer,an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitzdomain peptide, a monobody, or any combination thereof. In someembodiments, the capture probe and/or the anchor probe is conjugated tothe first solid support and/or the second solid support by a 1,3-dipolarcycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilicsubstitution reaction, a non-aldol type carbonyl reaction, an additionto carbon-carbon multiple bond, an oxidation reaction, a click reaction,or any combination thereof.

In some embodiments, the surface cellular target comprises acarbohydrate, a lipid, a protein, an extracellular protein, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an intracellular protein, or any combination thereof. Forexample, the surface cellular target can comprise a carbohydrate, alipid, a protein, or any combination thereof.

In some embodiments, the composition comprises a DNA polymerase (e.g., aKlenow Fragment) lacking at least one of 5′ to 3′ exonuclease activityand 3′ to 5′ exonuclease activity. In some embodiments, the compositioncomprises a reverse transcriptase, such as a viral reversetranscriptase. The composition can comprise a buffer, a cartridge, orboth.

In some embodiments, the composition comprises a plurality ofoligonucleotide barcodes, each oligonucleotide barcode of the pluralityof oligonucleotide barcodes comprises a target-binding region. Thetarget-binding region can comprise a poly(dA) region, a poly(dT) region,a random sequence, a gene-specific sequence, or any combination thereof.In some embodiments, the plurality of oligonucleotide barcodes eachcomprise a molecular label. The molecular label can comprise at least 6nucleotides. In some embodiments, at least 10 of the plurality ofoligonucleotide barcodes comprise different molecular label sequences.In some embodiments, the plurality of oligonucleotide barcodes areassociated with a third solid support. In some embodiments, theplurality of oligonucleotide barcodes each comprise a cell label. Insome embodiments, oligonucleotide barcodes of the plurality ofoligonucleotide barcodes associated with the same third solid supportcomprise the same cell label. In some embodiments, oligonucleotidebarcodes of the plurality of oligonucleotide barcodes associated withdifferent third solid supports comprise different cell labels.

In some embodiments, the first solid support, second solid support,and/or third solid support comprises a synthetic particle or a planarsurface. The at least one of the plurality of oligonucleotide barcodescan be immobilized or partially immobilized on the synthetic particle,or the at least one of the plurality of oligonucleotide barcodes isenclosed or partially enclosed in the synthetic particle. The syntheticparticle can be disruptable, e.g., a disruptable hydrogel particle.

In some embodiments, each of the plurality of oligonucleotide barcodescomprises a linker functional group, the synthetic particle comprises asolid support functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

In some embodiments, each of the plurality of anchor probes comprises alinker functional group, the synthetic particle comprises a solidsupport functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

In some embodiments, each of the plurality of capture probes comprises alinker functional group, the synthetic particle comprises a solidsupport functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

In some embodiments, the first solid support and/or the second solidsupport is sized and shaped to approximate a cell. In some embodiments,the first solid support and/or the second solid support has thedimensions of a cell. In some embodiments, the cell is a mammalian cell,a yeast cell, an insect cell, a plant cell, a bacterial cell, or anycombination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a non-limiting exemplary barcode.

FIG. 2 shows a non-limiting exemplary workflow of barcoding and digitalcounting.

FIG. 3 is a schematic illustration showing a non-limiting exemplaryprocess for generating an indexed library of targets barcoded at the3′-ends from a plurality of targets.

FIGS. 4A-4C show a schematic illustration of a non-limiting exemplaryworkflow for measurement of the number of copies of one or more secretedfactors secreted by a single cell.

FIGS. 5A-5C show a schematic illustration of a non-limiting exemplaryworkflow for simultaneous measurement of secreted factors and geneexpression of single cells.

FIG. 6 shows a non-limiting exemplary design of a secretedfactor-binding reagent specific oligonucleotide (antibodyoligonucleotide illustrated here) that is associated with a secretedfactor-binding reagent (antibody illustrated here).

FIGS. 7A-7D show a schematic illustration of a non-limiting exemplaryworkflow for simultaneous measurement of the number of copies of asecreted factor and a nucleic acid target.

FIG. 8 shows a schematic illustration of a non-limiting exemplaryworkflow of barcoding of a binding reagent oligonucleotide (antibodyoligonucleotide illustrated here) that is associated with a bindingreagent (antibody illustrated here).

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein and made part of the disclosure herein.

All patents, published patent applications, other publications, andsequences from GenBank, and other databases referred to herein areincorporated by reference in their entirety with respect to the relatedtechnology.

Quantifying small numbers of nucleic acids, for example messengerribonucleotide acid (mRNA) molecules, is clinically important fordetermining, for example, the genes that are expressed in a cell atdifferent stages of development or under different environmentalconditions. However, it can also be very challenging to determine theabsolute number of nucleic acid molecules (e.g., mRNA molecules),especially when the number of molecules is very small. One method todetermine the absolute number of molecules in a sample is digitalpolymerase chain reaction (PCR). Ideally, PCR produces an identical copyof a molecule at each cycle. However, PCR can have disadvantages suchthat each molecule replicates with a stochastic probability, and thisprobability varies by PCR cycle and gene sequence, resulting inamplification bias and inaccurate gene expression measurements.Stochastic barcodes with unique molecular labels (also referred to asmolecular indexes (MIs)) can be used to count the number of moleculesand correct for amplification bias. Stochastic barcoding, such as thePrecise™ assay (Cellular Research, Inc. (Palo Alto, Calif.)) andRhapsody™ assay (Becton, Dickinson and Company (Franklin Lakes, N.J.)),can correct for bias induced by PCR and library preparation steps byusing molecular labels (MLs) to label mRNAs during reverse transcription(RT).

The Precise™ assay can utilize a non-depleting pool of stochasticbarcodes with large number, for example 6561 to 65536, unique molecularlabel sequences on poly(T) oligonucleotides to hybridize to allpoly(A)-mRNAs in a sample during the RT step. A stochastic barcode cancomprise a universal PCR priming site. During RT, target gene moleculesreact randomly with stochastic barcodes. Each target molecule canhybridize to a stochastic barcode resulting to generate stochasticallybarcoded complementary ribonucleotide acid (cDNA) molecules). Afterlabeling, stochastically barcoded cDNA molecules from microwells of amicrowell plate can be pooled into a single tube for PCR amplificationand sequencing. Raw sequencing data can be analyzed to produce thenumber of reads, the number of stochastic barcodes with unique molecularlabel sequences, and the numbers of mRNA molecules.

Disclosed herein include methods of measuring the number of copies of asecreted factor secreted by a single cell. The method can comprise:contacting one or more single cells with a first plurality of firstsolid supports, the one or more single cells are capable of secreting aplurality of secreted factors, each first solid support comprises aplurality of capture probes capable of specifically binding to at leastone of the plurality of secreted factors secreted by a single cell. Themethod can comprise: contacting the first solid support with a pluralityof secreted factor-binding reagents each capable of specifically bindingto a secreted factor bound by a capture probe, each of the plurality ofsecreted factor-binding reagents comprises a secreted factor-bindingreagent specific oligonucleotide comprising a unique factor identifiersequence for the secreted factor-binding reagent. The method cancomprise: contacting a plurality of oligonucleotide barcodes with thesecreted factor-binding reagent specific oligonucleotides forhybridization, the oligonucleotide barcodes each comprise a firstmolecular label. The method can comprise: extending the plurality ofoligonucleotide barcodes hybridized to the secreted factor-bindingreagent specific oligonucleotides to generate a plurality of barcodedsecreted factor-binding reagent specific oligonucleotides eachcomprising a sequence complementary to at least a portion of the uniquefactor identifier sequence and the first molecular label. The method cancomprise: obtaining sequence information of the plurality of barcodedsecreted factor-binding reagent specific oligonucleotides, or productsthereof, to determine the number of copies of the at least one secretedfactor secreted by each of the one or more single cells. In someembodiments, the one or more single cells comprises T cells, B cells,tumor cells, myeloid cells, blood cells, normal cells, fetal cells,maternal cells, or a mixture thereof.

Disclosed herein include methods of measuring the number of copies of asecreted factor secreted by a single cell and the number of copies of anucleic acid target in a single cell. The method can comprise:contacting one or more single cells with a first plurality of secondsolid supports to form one or more single cells associated with a secondsolid support, the one or more single cells comprise a surface cellulartarget and copies of a nucleic acid target, the one or more single cellsare capable of secreting a plurality of secreted factors, each secondsolid support comprises a plurality of capture probes and a plurality ofanchor probes, each of the plurality of anchor probes is capable ofspecifically binding to the surface cellular target, and the captureprobe is capable of specifically binding to at least one of theplurality of secreted factors secreted by a single cell. The method cancomprise: contacting the one or more single cells associated with asecond solid support with a plurality of secreted factor-bindingreagents capable of specifically binding to a secreted factor bound by acapture probe, each of the plurality of secreted factor-binding reagentscomprises a secreted factor-binding reagent specific oligonucleotidecomprising a unique factor identifier sequence for the secretedfactor-binding reagent. The method can comprise: contacting a pluralityof oligonucleotide barcodes with the secreted factor-binding reagentspecific oligonucleotides and the copies of the nucleic acid target forhybridization, the oligonucleotide barcodes each comprise a firstmolecular label. The method can comprise: extending the plurality ofoligonucleotide barcodes hybridized to the copies of a nucleic acidtarget to generate a plurality of barcoded nucleic acid molecules eachcomprising a sequence complementary to at least a portion of the nucleicacid target and the first molecular label. The method can comprise:extending the plurality of oligonucleotide barcodes hybridized to thesecreted factor-binding reagent specific oligonucleotides to generate aplurality of barcoded secreted factor-binding reagent specificoligonucleotides each comprising a sequence complementary to at least aportion of the unique factor identifier sequence and the first molecularlabel. The method can comprise: obtaining sequence information of theplurality of barcoded nucleic acid molecules, or products thereof, todetermine the copy number of the nucleic acid target in each of the oneor more single cells. The method can comprise: obtaining sequenceinformation of the plurality of barcoded secreted factor-binding reagentspecific oligonucleotides, or products thereof, to determine the numberof copies of the at least one secreted factor secreted by each of theone or more single cells. In some embodiments, the one or more singlecells comprises T cells, B cells, tumor cells, myeloid cells, bloodcells, normal cells, fetal cells, maternal cells, or a mixture thereof.

Disclosed herein include compositions (e.g., kits). The composition cancomprise: a first solid support comprising a plurality of capture probescapable of specifically binding to at least one of a plurality ofsecreted factors secreted by a single cell; and a plurality of secretedfactor-binding reagents each capable of specifically binding to asecreted factor bound by a capture probe, each of the plurality ofsecreted factor-binding reagents comprises a secreted factor-bindingreagent specific oligonucleotide comprising a unique factor identifiersequence for the secreted factor-binding reagent.

Disclosed herein include compositions (e.g., kits). The composition cancomprise: a second solid support comprising a plurality of captureprobes and a plurality of anchor probes, each of the plurality of anchorprobes is capable of specifically binding to a surface cellular target,and the capture probe is capable of specifically binding to at least oneof a plurality of secreted factors secreted by a single cell; and aplurality of secreted factor-binding reagents each capable ofspecifically binding to a secreted factor bound by a capture probe, eachof the plurality of secreted factor-binding reagents comprises asecreted factor-binding reagent specific oligonucleotide comprising aunique factor identifier sequence for the secreted factor-bindingreagent.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. See, e.g., Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.1989). For purposes of the present disclosure, the following terms aredefined below.

As used herein, the term “adaptor” can mean a sequence to facilitateamplification or sequencing of associated nucleic acids. The associatednucleic acids can comprise target nucleic acids. The associated nucleicacids can comprise one or more of spatial labels, target labels, samplelabels, indexing label, or barcode sequences (e.g., molecular labels).The adaptors can be linear. The adaptors can be pre-adenylated adaptors.The adaptors can be double- or single-stranded. One or more adaptor canbe located on the 5′ or 3′ end of a nucleic acid. When the adaptorscomprise known sequences on the 5′ and 3′ ends, the known sequences canbe the same or different sequences. An adaptor located on the 5′ and/or3′ ends of a polynucleotide can be capable of hybridizing to one or moreoligonucleotides immobilized on a surface. An adaptor can, in someembodiments, comprise a universal sequence. A universal sequence can bea region of nucleotide sequence that is common to two or more nucleicacid molecules. The two or more nucleic acid molecules can also haveregions of different sequence. Thus, for example, the 5′ adaptors cancomprise identical and/or universal nucleic acid sequences and the 3′adaptors can comprise identical and/or universal sequences. A universalsequence that may be present in different members of a plurality ofnucleic acid molecules can allow the replication or amplification ofmultiple different sequences using a single universal primer that iscomplementary to the universal sequence. Similarly, at least one, two(e.g., a pair) or more universal sequences that may be present indifferent members of a collection of nucleic acid molecules can allowthe replication or amplification of multiple different sequences usingat least one, two (e.g., a pair) or more single universal primers thatare complementary to the universal sequences. Thus, a universal primerincludes a sequence that can hybridize to such a universal sequence. Thetarget nucleic acid sequence-bearing molecules may be modified to attachuniversal adaptors (e.g., non-target nucleic acid sequences) to one orboth ends of the different target nucleic acid sequences. The one ormore universal primers attached to the target nucleic acid can providesites for hybridization of universal primers. The one or more universalprimers attached to the target nucleic acid can be the same or differentfrom each other.

As used herein the term “associated” or “associated with” can mean thattwo or more species are identifiable as being co-located at a point intime. An association can mean that two or more species are or werewithin a similar container. An association can be an informaticsassociation. For example, digital information regarding two or morespecies can be stored and can be used to determine that one or more ofthe species were co-located at a point in time. An association can alsobe a physical association. In some embodiments, two or more associatedspecies are “tethered”, “attached”, or “immobilized” to one another orto a common solid or semisolid surface. An association may refer tocovalent or non-covalent means for attaching labels to solid orsemi-solid supports such as beads. An association may be a covalent bondbetween a target and a label. An association can comprise hybridizationbetween two molecules (such as a target molecule and a label).

As used herein, the term “complementary” can refer to the capacity forprecise pairing between two nucleotides. For example, if a nucleotide ata given position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position.Complementarity between two single-stranded nucleic acid molecules maybe “partial,” in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. A first nucleotide sequence can be said to be the“complement” of a second sequence if the first nucleotide sequence iscomplementary to the second nucleotide sequence. A first nucleotidesequence can be said to be the “reverse complement” of a secondsequence, if the first nucleotide sequence is complementary to asequence that is the reverse (i.e., the order of the nucleotides isreversed) of the second sequence. As used herein, a “complementary”sequence can refer to a “complement” or a “reverse complement” of asequence. It is understood from the disclosure that if a molecule canhybridize to another molecule it may be complementary, or partiallycomplementary, to the molecule that is hybridizing.

As used herein, the term “digital counting” can refer to a method forestimating a number of target molecules in a sample. Digital countingcan include the step of determining a number of unique labels that havebeen associated with targets in a sample. This methodology, which can bestochastic in nature, transforms the problem of counting molecules fromone of locating and identifying identical molecules to a series ofyes/no digital questions regarding detection of a set of predefinedlabels.

As used herein, the term “label” or “labels” can refer to nucleic acidcodes associated with a target within a sample. A label can be, forexample, a nucleic acid label. A label can be an entirely or partiallyamplifiable label. A label can be entirely or partially sequencablelabel. A label can be a portion of a native nucleic acid that isidentifiable as distinct. A label can be a known sequence. A label cancomprise a junction of nucleic acid sequences, for example a junction ofa native and non-native sequence. As used herein, the term “label” canbe used interchangeably with the terms, “index”, “tag,” or “label-tag.”Labels can convey information. For example, in various embodiments,labels can be used to determine an identity of a sample, a source of asample, an identity of a cell, and/or a target.

As used herein, the term “non-depleting reservoirs” can refer to a poolof barcodes (e.g., stochastic barcodes) made up of many differentlabels. A non-depleting reservoir can comprise large numbers ofdifferent barcodes such that when the non-depleting reservoir isassociated with a pool of targets each target is likely to be associatedwith a unique barcode. The uniqueness of each labeled target moleculecan be determined by the statistics of random choice, and depends on thenumber of copies of identical target molecules in the collectioncompared to the diversity of labels. The size of the resulting set oflabeled target molecules can be determined by the stochastic nature ofthe barcoding process, and analysis of the number of barcodes detectedthen allows calculation of the number of target molecules present in theoriginal collection or sample. When the ratio of the number of copies ofa target molecule present to the number of unique barcodes is low, thelabeled target molecules are highly unique (i.e., there is a very lowprobability that more than one target molecule will have been labeledwith a given label).

As used herein, the term “nucleic acid” refers to a polynucleotidesequence, or fragment thereof. A nucleic acid can comprise nucleotides.A nucleic acid can be exogenous or endogenous to a cell. A nucleic acidcan exist in a cell-free environment. A nucleic acid can be a gene orfragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA.A nucleic acid can comprise one or more analogs (e.g., altered backbone,sugar, or nucleobase). Some non-limiting examples of analogs include:5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos,locked nucleic acids, glycol nucleic acids, threose nucleic acids,dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g.,rhodamine or fluorescein linked to the sugar), thiol containingnucleotides, biotin linked nucleotides, fluorescent base analogs, CpGislands, methyl-7-guanosine, methylated nucleotides, inosine,thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.“Nucleic acid”, “polynucleotide, “target polynucleotide”, and “targetnucleic acid” can be used interchangeably.

A nucleic acid can comprise one or more modifications (e.g., a basemodification, a backbone modification), to provide the nucleic acid witha new or enhanced feature (e.g., improved stability). A nucleic acid cancomprise a nucleic acid affinity tag. A nucleoside can be a base-sugarcombination. The base portion of the nucleoside can be a heterocyclicbase. The two most common classes of such heterocyclic bases are thepurines and the pyrimidines. Nucleotides can be nucleosides that furtherinclude a phosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxylmoiety of the sugar. In forming nucleic acids, the phosphate groups cancovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound;however, linear compounds are generally suitable. In addition, linearcompounds may have internal nucleotide base complementarity and maytherefore fold in a manner as to produce a fully or partiallydouble-stranded compound. Within nucleic acids, the phosphate groups cancommonly be referred to as forming the internucleoside backbone of thenucleic acid. The linkage or backbone can be a 3′ to 5′ phosphodiesterlinkage.

A nucleic acid can comprise a modified backbone and/or modifiedinternucleoside linkages. Modified backbones can include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. Suitable modified nucleic acidbackbones containing a phosphorus atom therein can include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonate such as 3′ -alkylene phosphonates, 5′-alkylene phosphonates,chiral phosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs, and those havinginverted polarity wherein one or more internucleotide linkages is a 3′to 3′, a 5′ to 5′ or a 2′ to 2′ linkage.

A nucleic acid can comprise polynucleotide backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These can include those having morpholino linkages (formed in part fromthe sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; riboacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH₂ component parts.

A nucleic acid can comprise a nucleic acid mimetic. The term “mimetic”can be intended to include polynucleotides wherein only the furanosering or both the furanose ring and the internucleotide linkage arereplaced with non-furanose groups, replacement of only the furanose ringcan also be referred as being a sugar surrogate. The heterocyclic basemoiety or a modified heterocyclic base moiety can be maintained forhybridization with an appropriate target nucleic acid. One such nucleicacid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backboneof a polynucleotide can be replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleotides can beretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. The backbone in PNA compounds cancomprise two or more linked aminoethylglycine units which gives PNA anamide containing backbone. The heterocyclic base moieties can be bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone.

A nucleic acid can comprise a morpholino backbone structure. Forexample, a nucleic acid can comprise a 6-membered morpholino ring inplace of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagecan replace a phosphodiester linkage.

A nucleic acid can comprise linked morpholino units (e.g., morpholinonucleic acid) having heterocyclic bases attached to the morpholino ring.Linking groups can link the morpholino monomeric units in a morpholinonucleic acid. Non-ionic morpholino-based oligomeric compounds can haveless undesired interactions with cellular proteins. Morpholino-basedpolynucleotides can be nonionic mimics of nucleic acids. A variety ofcompounds within the morpholino class can be joined using differentlinking groups. A further class of polynucleotide mimetic can bereferred to as cyclohexenyl nucleic acids (CeNA). The furanose ringnormally present in a nucleic acid molecule can be replaced with acyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can beprepared and used for oligomeric compound synthesis usingphosphoramidite chemistry. The incorporation of CeNA monomers into anucleic acid chain can increase the stability of a DNA/RNA hybrid. CeNAoligoadenylates can form complexes with nucleic acid complements withsimilar stability to the native complexes. A further modification caninclude Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group islinked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. Thelinkage can be a methylene (—CH₂), group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNA and LNA analogs can displayvery high duplex thermal stabilities with complementary nucleic acid(Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation andgood solubility properties.

A nucleic acid may also include nucleobase (often referred to simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases can include the purine bases, (e.g., adenine (A)and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine(C) and uracil (U)). Modified nucleobases can include other syntheticand natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH3) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modifiednucleobases can include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one),G-clamps such as a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo[2,3-d]pyrimidin-2-one).

As used herein, the term “sample” can refer to a composition comprisingtargets. Suitable samples for analysis by the disclosed methods,devices, and systems include cells, tissues, organs, or organisms.

As used herein, the term “sampling device” or “device” can refer to adevice which may take a section of a sample and/or place the section ona substrate. A sample device can refer to, for example, a fluorescenceactivated cell sorting (FACS) machine, a cell sorter machine, a biopsyneedle, a biopsy device, a tissue sectioning device, a microfluidicdevice, a blade grid, and/or a microtome.

As used herein, the term “solid support” can refer to discrete solid orsemi-solid surfaces to which a plurality of barcodes (e.g., stochasticbarcodes) may be attached. A solid support may encompass any type ofsolid, porous, or hollow sphere, ball, bearing, cylinder, or othersimilar configuration composed of plastic, ceramic, metal, or polymericmaterial (e.g., hydrogel) onto which a nucleic acid may be immobilized(e.g., covalently or non-covalently). A solid support may comprise adiscrete particle that may be spherical (e.g., microspheres) or have anon-spherical or irregular shape, such as cubic, cuboid, pyramidal,cylindrical, conical, oblong, or disc-shaped, and the like. A bead canbe non-spherical in shape. A plurality of solid supports spaced in anarray may not comprise a substrate. A solid support may be usedinterchangeably with the term “bead.”

As used herein, the term “stochastic barcode” can refer to apolynucleotide sequence comprising labels of the present disclosure. Astochastic barcode can be a polynucleotide sequence that can be used forstochastic barcoding. Stochastic barcodes can be used to quantifytargets within a sample. Stochastic barcodes can be used to control forerrors which may occur after a label is associated with a target. Forexample, a stochastic barcode can be used to assess amplification orsequencing errors. A stochastic barcode associated with a target can becalled a stochastic barcode-target or stochastic barcode-tag-target.

As used herein, the term “gene-specific stochastic barcode” can refer toa polynucleotide sequence comprising labels and a target-binding regionthat is gene-specific. A stochastic barcode can be a polynucleotidesequence that can be used for stochastic barcoding. Stochastic barcodescan be used to quantify targets within a sample. Stochastic barcodes canbe used to control for errors which may occur after a label isassociated with a target. For example, a stochastic barcode can be usedto assess amplification or sequencing errors. A stochastic barcodeassociated with a target can be called a stochastic barcode-target orstochastic barcode-tag-target.

As used herein, the term “stochastic barcoding” can refer to the randomlabeling (e.g., barcoding) of nucleic acids. Stochastic barcoding canutilize a recursive Poisson strategy to associate and quantify labelsassociated with targets. As used herein, the term “stochastic barcoding”can be used interchangeably with “stochastic labeling.”

As used here, the term “target” can refer to a composition which can beassociated with a barcode (e.g., a stochastic barcode). Exemplarysuitable targets for analysis by the disclosed methods, devices, andsystems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, andthe like. Targets can be single or double stranded. In some embodiments,targets can be proteins, peptides, or polypeptides. In some embodiments,targets are lipids. As used herein, “target” can be used interchangeablywith “species.”

As used herein, the term “reverse transcriptases” can refer to a groupof enzymes having reverse transcriptase activity (i.e., that catalyzesynthesis of DNA from an RNA template). In general, such enzymesinclude, but are not limited to, retroviral reverse transcriptase,retrotransposon reverse transcriptase, retroplasmid reversetranscriptases, retron reverse transcriptases, bacterial reversetranscriptases, group II intron-derived reverse transcriptase, andmutants, variants or derivatives thereof. Non-retroviral reversetranscriptases include non-LTR retrotransposon reverse transcriptases,retroplasmid reverse transcriptases, retron reverse transcriptases, andgroup II intron reverse transcriptases. Examples of group II intronreverse transcriptases include the Lactococcus lactis LI.LtrB intronreverse transcriptase, the Thermosynechococcus elongatus TeI4c intronreverse transcriptase, or the Geobacillus stearothermophilus GsI-IICintron reverse transcriptase. Other classes of reverse transcriptasescan include many classes of non-retroviral reverse transcriptases (i.e.,retrons, group II introns, and diversity-generating retroelements amongothers).

The terms “universal adaptor primer,” “universal primer adaptor” or“universal adaptor sequence” are used interchangeably to refer to anucleotide sequence that can be used to hybridize to barcodes (e.g.,stochastic barcodes) to generate gene-specific barcodes. A universaladaptor sequence can, for example, be a known sequence that is universalacross all barcodes used in methods of the disclosure. For example, whenmultiple targets are being labeled using the methods disclosed herein,each of the target-specific sequences may be linked to the sameuniversal adaptor sequence. In some embodiments, more than one universaladaptor sequences may be used in the methods disclosed herein. Forexample, when multiple targets are being labeled using the methodsdisclosed herein, at least two of the target-specific sequences arelinked to different universal adaptor sequences. A universal adaptorprimer and its complement may be included in two oligonucleotides, oneof which comprises a target-specific sequence and the other comprises abarcode. For example, a universal adaptor sequence may be part of anoligonucleotide comprising a target-specific sequence to generate anucleotide sequence that is complementary to a target nucleic acid. Asecond oligonucleotide comprising a barcode and a complementary sequenceof the universal adaptor sequence may hybridize with the nucleotidesequence and generate a target-specific barcode (e.g., a target-specificstochastic barcode). In some embodiments, a universal adaptor primer hasa sequence that is different from a universal PCR primer used in themethods of this disclosure.

Barcodes

Barcoding, such as stochastic barcoding, has been described in, forexample, Fu et al., Proc Natl Acad Sci U.S.A., 2011 May 31,108(22):9026-31; U.S. Patent Application Publication No. US2011/0160078;Fan et al., Science, 2015 Feb. 6, 347(6222):1258367; US PatentApplication Publication No. US2015/0299784; and PCT ApplicationPublication No. WO2015/031691; the content of each of these, includingany supporting or supplemental information or material, is incorporatedherein by reference in its entirety. In some embodiments, the barcodedisclosed herein can be a stochastic barcode which can be apolynucleotide sequence that may be used to stochastically label (e.g.,barcode, tag) a target. Barcodes can be referred to stochastic barcodesif the ratio of the number of different barcode sequences of thestochastic barcodes and the number of occurrence of any of the targetsto be labeled can be, or be about, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or arange between any two of these values. A target can be an mRNA speciescomprising mRNA molecules with identical or nearly identical sequences.Barcodes can be referred to as stochastic barcodes if the ratio of thenumber of different barcode sequences of the stochastic barcodes and thenumber of occurrence of any of the targets to be labeled is at least, oris at most, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1,60:1, 70:1, 80:1, 90:1, or 100:1. Barcode sequences of stochasticbarcodes can be referred to as molecular labels.

A barcode, for example a stochastic barcode, can comprise one or morelabels. Exemplary labels can include a universal label, a cell label, abarcode sequence (e.g., a molecular label), a sample label, a platelabel, a spatial label, and/or a pre-spatial label. FIG. 1 illustratesan exemplary barcode 104 with a spatial label. The barcode 104 cancomprise a 5′ amine that may link the barcode to a solid support 105.The barcode can comprise a universal label, a dimension label, a spatiallabel, a cell label, and/or a molecular label. The order of differentlabels (including but not limited to the universal label, the dimensionlabel, the spatial label, the cell label, and the molecule label) in thebarcode can vary. For example, as shown in FIG. 1, the universal labelmay be the 5′-most label, and the molecular label may be the 3′-mostlabel. The spatial label, dimension label, and the cell label may be inany order. In some embodiments, the universal label, the spatial label,the dimension label, the cell label, and the molecular label are in anyorder. The barcode can comprise a target-binding region. Thetarget-binding region can interact with a target (e.g., target nucleicacid, RNA, mRNA, DNA) in a sample. For example, a target-binding regioncan comprise an oligo(dT) sequence which can interact with poly(A) tailsof mRNAs. In some instances, the labels of the barcode (e.g., universallabel, dimension label, spatial label, cell label, and barcode sequence)may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 or more nucleotides.

A label, for example the cell label, can comprise a unique set ofnucleic acid sub-sequences of defined length, e.g., seven nucleotideseach (equivalent to the number of bits used in some Hamming errorcorrection codes), which can be designed to provide error correctioncapability. The set of error correction sub-sequences comprise sevennucleotide sequences can be designed such that any pairwise combinationof sequences in the set exhibits a defined “genetic distance” (or numberof mismatched bases), for example, a set of error correctionsub-sequences can be designed to exhibit a genetic distance of threenucleotides. In this case, review of the error correction sequences inthe set of sequence data for labeled target nucleic acid molecules(described more fully below) can allow one to detect or correctamplification or sequencing errors. In some embodiments, the length ofthe nucleic acid sub-sequences used for creating error correction codescan vary, for example, they can be, or be about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 31, 40, 50, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, nucleic acidsub-sequences of other lengths can be used for creating error correctioncodes.

The barcode can comprise a target-binding region. The target-bindingregion can interact with a target in a sample. The target can be, orcomprise, ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs,small interfering RNAs (siRNAs), RNA degradation products, RNAs eachcomprising a poly(A) tail, or any combination thereof. In someembodiments, the plurality of targets can include deoxyribonucleic acids(DNAs).

In some embodiments, a target-binding region can comprise an oligo(dT)sequence which can interact with poly(A) tails of mRNAs. One or more ofthe labels of the barcode (e.g., the universal label, the dimensionlabel, the spatial label, the cell label, and the barcode sequences(e.g., molecular label)) can be separated by a spacer from another oneor two of the remaining labels of the barcode. The spacer can be, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, or more nucleotides. In some embodiments, none of the labelsof the barcode is separated by spacer.

Universal Labels

A barcode can comprise one or more universal labels. In someembodiments, the one or more universal labels can be the same for allbarcodes in the set of barcodes attached to a given solid support. Insome embodiments, the one or more universal labels can be the same forall barcodes attached to a plurality of beads. In some embodiments, auniversal label can comprise a nucleic acid sequence that is capable ofhybridizing to a sequencing primer. Sequencing primers can be used forsequencing barcodes comprising a universal label. Sequencing primers(e.g., universal sequencing primers) can comprise sequencing primersassociated with high-throughput sequencing platforms. In someembodiments, a universal label can comprise a nucleic acid sequence thatis capable of hybridizing to a PCR primer. In some embodiments, theuniversal label can comprise a nucleic acid sequence that is capable ofhybridizing to a sequencing primer and a PCR primer. The nucleic acidsequence of the universal label that is capable of hybridizing to asequencing or PCR primer can be referred to as a primer binding site. Auniversal label can comprise a sequence that can be used to initiatetranscription of the barcode. A universal label can comprise a sequencethat can be used for extension of the barcode or a region within thebarcode. A universal label can be, or be about, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, or a number or a range between any two ofthese values, nucleotides in length. For example, a universal label cancomprise at least about 10 nucleotides. A universal label can be atleast, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 200, or 300 nucleotides in length. In some embodiments, a cleavablelinker or modified nucleotide can be part of the universal labelsequence to enable the barcode to be cleaved off from the support.

Dimension Labels

A barcode can comprise one or more dimension labels. In someembodiments, a dimension label can comprise a nucleic acid sequence thatprovides information about a dimension in which the labeling (e.g.,stochastic labeling) occurred. For example, a dimension label canprovide information about the time at which a target was barcoded. Adimension label can be associated with a time of barcoding (e.g.,stochastic barcoding) in a sample. A dimension label can be activated atthe time of labeling. Different dimension labels can be activated atdifferent times. The dimension label provides information about theorder in which targets, groups of targets, and/or samples were barcoded.For example, a population of cells can be barcoded at the GO phase ofthe cell cycle. The cells can be pulsed again with barcodes (e.g.,stochastic barcodes) at the G1 phase of the cell cycle. The cells can bepulsed again with barcodes at the S phase of the cell cycle, and so on.Barcodes at each pulse (e.g., each phase of the cell cycle), cancomprise different dimension labels. In this way, the dimension labelprovides information about which targets were labelled at which phase ofthe cell cycle. Dimension labels can interrogate many differentbiological times. Exemplary biological times can include, but are notlimited to, the cell cycle, transcription (e.g., transcriptioninitiation), and transcript degradation. In another example, a sample(e.g., a cell, a population of cells) can be labeled before and/or aftertreatment with a drug and/or therapy. The changes in the number ofcopies of distinct targets can be indicative of the sample's response tothe drug and/or therapy.

A dimension label can be activatable. An activatable dimension label canbe activated at a specific time point. The activatable label can be, forexample, constitutively activated (e.g., not turned off). Theactivatable dimension label can be, for example, reversibly activated(e.g., the activatable dimension label can be turned on and turned off).The dimension label can be, for example, reversibly activatable at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The dimension label can bereversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more times. In some embodiments, the dimension label can beactivated with fluorescence, light, a chemical event (e.g., cleavage,ligation of another molecule, addition of modifications (e.g.,pegylated, sumoylated, acetylated, methylated, deacetylated,demethylated), a photochemical event (e.g., photocaging), andintroduction of a non-natural nucleotide.

The dimension label can, in some embodiments, be identical for allbarcodes (e.g., stochastic barcodes) attached to a given solid support(e.g., a bead), but different for different solid supports (e.g.,beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%,99% or 100%, of barcodes on the same solid support can comprise the samedimension label. In some embodiments, at least 60% of barcodes on thesame solid support can comprise the same dimension label. In someembodiments, at least 95% of barcodes on the same solid support cancomprise the same dimension label.

There can be as many as 10⁶ or more unique dimension label sequencesrepresented in a plurality of solid supports (e.g., beads). A dimensionlabel can be, or be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or a number or a range between any two of these values, nucleotidesin length. A dimension label can be at least, or be at most, 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300, nucleotides inlength. A dimension label can comprise between about 5 to about 200nucleotides. A dimension label can comprise between about 10 to about150 nucleotides. A dimension label can comprise between about 20 toabout 125 nucleotides in length.

Spatial Labels

A barcode can comprise one or more spatial labels. In some embodiments,a spatial label can comprise a nucleic acid sequence that providesinformation about the spatial orientation of a target molecule which isassociated with the barcode. A spatial label can be associated with acoordinate in a sample. The coordinate can be a fixed coordinate. Forexample, a coordinate can be fixed in reference to a substrate. Aspatial label can be in reference to a two or three-dimensional grid. Acoordinate can be fixed in reference to a landmark. The landmark can beidentifiable in space. A landmark can be a structure which can beimaged. A landmark can be a biological structure, for example ananatomical landmark. A landmark can be a cellular landmark, for instancean organelle. A landmark can be a non-natural landmark such as astructure with an identifiable identifier such as a color code, barcode, magnetic property, fluorescents, radioactivity, or a unique sizeor shape. A spatial label can be associated with a physical partition(e.g., A well, a container, or a droplet). In some embodiments, multiplespatial labels are used together to encode one or more positions inspace.

The spatial label can be identical for all barcodes attached to a givensolid support (e.g., a bead), but different for different solid supports(e.g., beads). In some embodiments, the percentage of barcodes on thesame solid support comprising the same spatial label can be, or beabout, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or arange between any two of these values. In some embodiments, thepercentage of barcodes on the same solid support comprising the samespatial label can be at least, or be at most, 60%, 70%, 80%, 85%, 90%,95%, 97%, 99%, or 100%. In some embodiments, at least 60% of barcodes onthe same solid support can comprise the same spatial label. In someembodiments, at least 95% of barcodes on the same solid support cancomprise the same spatial label.

There can be as many as 10⁶ or more unique spatial label sequencesrepresented in a plurality of solid supports (e.g., beads). A spatiallabel can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, or a number or a range between any two of these values,nucleotides in length. A spatial label can be at least or at most 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300nucleotides in length. A spatial label can comprise between about 5 toabout 200 nucleotides. A spatial label can comprise between about 10 toabout 150 nucleotides. A spatial label can comprise between about 20 toabout 125 nucleotides in length.

Cell Labels

A barcode (e.g., a stochastic barcode) can comprise one or more celllabels. In some embodiments, a cell label can comprise a nucleic acidsequence that provides information for determining which target nucleicacid originated from which cell. In some embodiments, the cell label isidentical for all barcodes attached to a given solid support (e.g., abead), but different for different solid supports (e.g., beads). In someembodiments, the percentage of barcodes on the same solid supportcomprising the same cell label can be, or be about 60%, 70%, 80%, 85%,90%, 95%, 97%, 99%, 100%, or a number or a range between any two ofthese values. In some embodiments, the percentage of barcodes on thesame solid support comprising the same cell label can be, or be about60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least60% of barcodes on the same solid support can comprise the same celllabel. As another example, at least 95% of barcodes on the same solidsupport can comprise the same cell label.

There can be as many as 10⁶ or more unique cell label sequencesrepresented in a plurality of solid supports (e.g., beads). A cell labelcan be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,or a number or a range between any two of these values, nucleotides inlength. A cell label can be at least, or be at most, 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.For example, a cell label can comprise between about 5 to about 200nucleotides. As another example, a cell label can comprise between about10 to about 150 nucleotides. As yet another example, a cell label cancomprise between about 20 to about 125 nucleotides in length.

Barcode Sequences

A barcode can comprise one or more barcode sequences. In someembodiments, a barcode sequence can comprise a nucleic acid sequencethat provides identifying information for the specific type of targetnucleic acid species hybridized to the barcode. A barcode sequence cancomprise a nucleic acid sequence that provides a counter (e.g., thatprovides a rough approximation) for the specific occurrence of thetarget nucleic acid species hybridized to the barcode (e.g.,target-binding region).

In some embodiments, a diverse set of barcode sequences are attached toa given solid support (e.g., a bead). In some embodiments, there can be,or be about, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or arange between any two of these values, unique molecular label sequences.For example, a plurality of barcodes can comprise about 6561 barcodessequences with distinct sequences. As another example, a plurality ofbarcodes can comprise about 65536 barcode sequences with distinctsequences. In some embodiments, there can be at least, or be at most,10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, unique barcode sequences. Theunique molecular label sequences can be attached to a given solidsupport (e.g., a bead). In some embodiments, the unique molecular labelsequence is partially or entirely encompassed by a particle (e.g., ahydrogel bead).

The length of a barcode can be different in different implementations.For example, a barcode can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, or a number or a range between any two of thesevalues, nucleotides in length. As another example, a barcode can be atleast, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 200, or 300 nucleotides in length.

Molecular Labels

A barcode (e.g., a stochastic barcode) can comprise one or moremolecular labels. Molecular labels can include barcode sequences. Insome embodiments, a molecular label can comprise a nucleic acid sequencethat provides identifying information for the specific type of targetnucleic acid species hybridized to the barcode. A molecular label cancomprise a nucleic acid sequence that provides a counter for thespecific occurrence of the target nucleic acid species hybridized to thebarcode (e.g., target-binding region).

In some embodiments, a diverse set of molecular labels are attached to agiven solid support (e.g., a bead). In some embodiments, there can be,or be about, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or arange between any two of these values, of unique molecular labelsequences. For example, a plurality of barcodes can comprise about 6561molecular labels with distinct sequences. As another example, aplurality of barcodes can comprise about 65536 molecular labels withdistinct sequences. In some embodiments, there can be at least, or be atmost, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, unique molecular labelsequences. Barcodes with unique molecular label sequences can beattached to a given solid support (e.g., a bead).

For barcoding (e.g., stochastic barcoding) using a plurality ofstochastic barcodes, the ratio of the number of different molecularlabel sequences and the number of occurrence of any of the targets canbe, or be about, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1,50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or a range between anytwo of these values. A target can be an mRNA species comprising mRNAmolecules with identical or nearly identical sequences. In someembodiments, the ratio of the number of different molecular labelsequences and the number of occurrence of any of the targets is atleast, or is at most, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1,50:1, 60:1, 70:1, 80:1, 90:1, or 100:1

A molecular label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, or a number or a range between any two of thesevalues, nucleotides in length. A molecular label can be at least, or beat most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or300 nucleotides in length.

Target-Binding Region

A barcode can comprise one or more target binding regions, such ascapture probes. In some embodiments, a target-binding region canhybridize with a target of interest. In some embodiments, the targetbinding regions can comprise a nucleic acid sequence that hybridizesspecifically to a target (e.g., target nucleic acid, target molecule,e.g., a cellular nucleic acid to be analyzed), for example to a specificgene sequence. In some embodiments, a target binding region can comprisea nucleic acid sequence that can attach (e.g., hybridize) to a specificlocation of a specific target nucleic acid. In some embodiments, thetarget binding region can comprise a nucleic acid sequence that iscapable of specific hybridization to a restriction enzyme site overhang(e.g., an EcoRI sticky-end overhang). The barcode can then ligate to anynucleic acid molecule comprising a sequence complementary to therestriction site overhang.

In some embodiments, a target binding region can comprise a non-specifictarget nucleic acid sequence. A non-specific target nucleic acidsequence can refer to a sequence that can bind to multiple targetnucleic acids, independent of the specific sequence of the targetnucleic acid. For example, target binding region can comprise a randommultimer sequence, a poly(dA) sequence, a poly(dT) sequence, a poly(dG)sequence, a poly(dC) sequence, or a combination thereof. For example,the target binding region can be an oligo(dT) sequence that hybridizesto the poly(A) tail on mRNA molecules. A random multimer sequence canbe, for example, a random dimer, trimer, quatramer, pentamer, hexamer,septamer, octamer, nonamer, decamer, or higher multimer sequence of anylength. In some embodiments, the target binding region is the same forall barcodes attached to a given bead. In some embodiments, the targetbinding regions for the plurality of barcodes attached to a given beadcan comprise two or more different target binding sequences. A targetbinding region can be, or be about, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or a number or a range between any two of these values, nucleotidesin length. A target binding region can be at most about 5, 10, 15, 20,25, 30, 35, 40, 45, 50 or more nucleotides in length. For example, anmRNA molecule can be reverse transcribed using a reverse transcriptase,such as Moloney murine leukemia virus (MMLV) reverse transcriptase, togenerate a cDNA molecule with a poly(dC) tail. A barcode can include atarget binding region with a poly(dG) tail. Upon base pairing betweenthe poly(dG) tail of the barcode and the poly(dC) tail of the cDNAmolecule, the reverse transcriptase switches template strands, fromcellular RNA molecule to the barcode, and continues replication to the5′ end of the barcode. By doing so, the resulting cDNA molecule containsthe sequence of the barcode (such as the molecular label) on the 3′ endof the cDNA molecule.

In some embodiments, a target-binding region can comprise an oligo(dT)which can hybridize with mRNAs comprising polyadenylated ends. Atarget-binding region can be gene-specific. For example, atarget-binding region can be configured to hybridize to a specificregion of a target. A target-binding region can be, or be about, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26 27, 28, 29, 30, or a number or a range between any two ofthese values, nucleotides in length. A target-binding region can be atleast, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30,nucleotides in length. A target-binding region can be about 5-30nucleotides in length. When a barcode comprises a gene-specifictarget-binding region, the barcode can be referred to herein as agene-specific barcode.

Orientation Property

A stochastic barcode (e.g., a stochastic barcode) can comprise one ormore orientation properties which can be used to orient (e.g., align)the barcodes. A barcode can comprise a moiety for isoelectric focusing.Different barcodes can comprise different isoelectric focusing points.When these barcodes are introduced to a sample, the sample can undergoisoelectric focusing in order to orient the barcodes into a known way.In this way, the orientation property can be used to develop a known mapof barcodes in a sample. Exemplary orientation properties can include,electrophoretic mobility (e.g., based on size of the barcode),isoelectric point, spin, conductivity, and/or self-assembly. Forexample, barcodes with an orientation property of self-assembly, canself-assemble into a specific orientation (e.g., nucleic acidnanostructure) upon activation.

Affinity Property

A barcode (e.g., a stochastic barcode) can comprise one or more affinityproperties. For example, a spatial label can comprise an affinityproperty. An affinity property can include a chemical and/or biologicalmoiety that can facilitate binding of the barcode to another entity(e.g., cell receptor). For example, an affinity property can comprise anantibody, for example, an antibody specific for a specific moiety (e.g.,receptor) on a sample. In some embodiments, the antibody can guide thebarcode to a specific cell type or molecule. Targets at and/or near thespecific cell type or molecule can be labeled (e.g., stochasticallylabeled). The affinity property can, in some embodiments, providespatial information in addition to the nucleotide sequence of thespatial label because the antibody can guide the barcode to a specificlocation. The antibody can be a therapeutic antibody, for example amonoclonal antibody or a polyclonal antibody. The antibody can behumanized or chimeric. The antibody can be a naked antibody or a fusionantibody.

The antibody can be a full-length (i.e., naturally occurring or formedby normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., specifically binding) portion of an immunoglobulinmolecule, like an antibody fragment.

The antibody fragment can be, for example, a portion of an antibody suchas F(ab′)2, Fab′, Fab, Fv, sFv and the like. In some embodiments, theantibody fragment can bind with the same antigen that is recognized bythe full-length antibody. The antibody fragment can include isolatedfragments consisting of the variable regions of antibodies, such as the“Fv” fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”). Exemplary antibodies can include, but are not limited to,antibodies for cancer cells, antibodies for viruses, antibodies thatbind to cell surface receptors (CD8, CD34, CD45), and therapeuticantibodies.

Universal Adaptor Primer

A barcode can comprise one or more universal adaptor primers. Forexample, a gene-specific barcode, such as a gene-specific stochasticbarcode, can comprise a universal adaptor primer. A universal adaptorprimer can refer to a nucleotide sequence that is universal across allbarcodes. A universal adaptor primer can be used for buildinggene-specific barcodes. A universal adaptor primer can be, or be about,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26 27, 28, 29, 30, or a number or a range betweenany two of these nucleotides in length. A universal adaptor primer canbe at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30nucleotides in length. A universal adaptor primer can be from 5-30nucleotides in length.

Linker

When a barcode comprises more than one of a type of label (e.g., morethan one cell label or more than one barcode sequence, such as onemolecular label), the labels may be interspersed with a linker labelsequence. A linker label sequence can be at least about 5, 10, 15, 20,25, 30, 35, 40, 45, 50 or more nucleotides in length. A linker labelsequence can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 ormore nucleotides in length. In some instances, a linker label sequenceis 12 nucleotides in length. A linker label sequence can be used tofacilitate the synthesis of the barcode. The linker label can comprisean error-correcting (e.g., Hamming) code.

Solid Supports

Barcodes, such as stochastic barcodes, disclosed herein can, in someembodiments, be associated with a solid support. The solid support canbe, for example, a synthetic particle. In some embodiments, some or allof the barcode sequences, such as molecular labels for stochasticbarcodes (e.g., the first barcode sequences) of a plurality of barcodes(e.g., the first plurality of barcodes) on a solid support differ by atleast one nucleotide. The cell labels of the barcodes on the same solidsupport can be the same. The cell labels of the barcodes on differentsolid supports can differ by at least one nucleotide. For example, firstcell labels of a first plurality of barcodes on a first solid supportcan have the same sequence, and second cell labels of a second pluralityof barcodes on a second solid support can have the same sequence. Thefirst cell labels of the first plurality of barcodes on the first solidsupport and the second cell labels of the second plurality of barcodeson the second solid support can differ by at least one nucleotide. Acell label can be, for example, about 5-20 nucleotides long. A barcodesequence can be, for example, about 5-20 nucleotides long. The syntheticparticle can be, for example, a bead.

The bead can be, for example, a silica gel bead, a controlled pore glassbead, a magnetic bead, a Dynabead, a Sephadex/Sepharose bead, acellulose bead, a polystyrene bead, or any combination thereof. The beadcan comprise a material such as polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, Sepharose, cellulose, nylon, silicone, or anycombination thereof.

In some embodiments, the bead can be a polymeric bead, for example adeformable bead or a gel bead, functionalized with barcodes orstochastic barcodes (such as gel beads from 10X Genomics (San Francisco,Calif.). In some implementation, a gel bead can comprise a polymer basedgels. Gel beads can be generated, for example, by encapsulating one ormore polymeric precursors into droplets. Upon exposure of the polymericprecursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)),a gel bead may be generated.

In some embodiments, the particle can be disruptable (e.g., dissolvable,degradable). For example, the polymeric bead can dissolve, melt, ordegrade, for example, under a desired condition. The desired conditioncan include an environmental condition. The desired condition may resultin the polymeric bead dissolving, melting, or degrading in a controlledmanner. A gel bead may dissolve, melt, or degrade due to a chemicalstimulus, a physical stimulus, a biological stimulus, a thermalstimulus, a magnetic stimulus, an electric stimulus, a light stimulus,or any combination thereof.

Analytes and/or reagents, such as oligonucleotide barcodes, for example,may be coupled/immobilized to the interior surface of a gel bead (e.g.,the interior accessible via diffusion of an oligonucleotide barcodeand/or materials used to generate an oligonucleotide barcode) and/or theouter surface of a gel bead or any other microcapsule described herein.Coupling/immobilization may be via any form of chemical bonding (e.g.,covalent bond, ionic bond) or physical phenomena (e.g., Van der Waalsforces, dipole-dipole interactions, etc.). In some embodiments,coupling/immobilization of a reagent to a gel bead or any othermicrocapsule described herein may be reversible, such as, for example,via a labile moiety (e.g., via a chemical cross-linker, includingchemical cross-linkers described herein). Upon application of astimulus, the labile moiety may be cleaved and the immobilized reagentset free. In some embodiments, the labile moiety is a disulfide bond.For example, in the case where an oligonucleotide barcode is immobilizedto a gel bead via a disulfide bond, exposure of the disulfide bond to areducing agent can cleave the disulfide bond and free theoligonucleotide barcode from the bead. The labile moiety may be includedas part of a gel bead or microcapsule, as part of a chemical linker thatlinks a reagent or analyte to a gel bead or microcapsule, and/or as partof a reagent or analyte. In some embodiments, at least one barcode ofthe plurality of barcodes can be immobilized on the particle, partiallyimmobilized on the particle, enclosed in the particle, partiallyenclosed in the particle, or any combination thereof.

In some embodiments, a gel bead can comprise a wide range of differentpolymers including but not limited to: polymers, heat sensitivepolymers, photosensitive polymers, magnetic polymers, pH sensitivepolymers, salt-sensitive polymers, chemically sensitive polymers,polyelectrolytes, polysaccharides, peptides, proteins, and/or plastics.Polymers may include but are not limited to materials such aspoly(N-isopropylacrylamide) (PNIPAAm), poly(styrene sulfonate) (PSS),poly(allyl amine) (PAAm), poly(acrylic acid) (PAA), poly(ethylene imine)(PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrolle)(PPy), poly(vinylpyrrolidone) (PVPON), poly(vinyl pyridine) (PVP),poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA),polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthaladehyde)(PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL),poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).

Numerous chemical stimuli can be used to trigger the disruption,dissolution, or degradation of the beads. Examples of these chemicalchanges may include, but are not limited to pH-mediated changes to thebead wall, disintegration of the bead wall via chemical cleavage ofcrosslink bonds, triggered depolymerization of the bead wall, and beadwall switching reactions. Bulk changes may also be used to triggerdisruption of the beads.

Bulk or physical changes to the microcapsule through various stimulialso offer many advantages in designing capsules to release reagents.Bulk or physical changes occur on a macroscopic scale, in which beadrupture is the result of mechano-physical forces induced by a stimulus.These processes may include, but are not limited to pressure inducedrupture, bead wall melting, or changes in the porosity of the bead wall.

Biological stimuli may also be used to trigger disruption, dissolution,or degradation of beads. Generally, biological triggers resemblechemical triggers, but many examples use biomolecules, or moleculescommonly found in living systems such as enzymes, peptides, saccharides,fatty acids, nucleic acids and the like. For example, beads may comprisepolymers with peptide cross-links that are sensitive to cleavage byspecific proteases. More specifically, one example may comprise amicrocapsule comprising GFLGK peptide cross links. Upon addition of abiological trigger such as the protease Cathepsin B, the peptide crosslinks of the shell well are cleaved and the contents of the beads arereleased. In other cases, the proteases may be heat-activated. Inanother example, beads comprise a shell wall comprising cellulose.Addition of the hydrolytic enzyme chitosan serves as biologic triggerfor cleavage of cellulosic bonds, depolymerization of the shell wall,and release of its inner contents.

The beads may also be induced to release their contents upon theapplication of a thermal stimulus. A change in temperature can cause avariety changes to the beads. A change in heat may cause melting of abead such that the bead wall disintegrates. In other cases, the heat mayincrease the internal pressure of the inner components of the bead suchthat the bead ruptures or explodes. In still other cases, the heat maytransform the bead into a shrunken dehydrated state. The heat may alsoact upon heat-sensitive polymers within the wall of a bead to causedisruption of the bead.

Inclusion of magnetic nanoparticles to the bead wall of microcapsulesmay allow triggered rupture of the beads as well as guide the beads inan array. A device of this disclosure may comprise magnetic beads foreither purpose. In one example, incorporation of Fe₃O₄ nanoparticlesinto polyelectrolyte containing beads triggers rupture in the presenceof an oscillating magnetic field stimulus.

A bead may also be disrupted, dissolved, or degraded as the result ofelectrical stimulation. Similar to magnetic particles described in theprevious section, electrically sensitive beads can allow for bothtriggered rupture of the beads as well as other functions such asalignment in an electric field, electrical conductivity or redoxreactions. In one example, beads containing electrically sensitivematerial are aligned in an electric field such that release of innerreagents can be controlled. In other examples, electrical fields mayinduce redox reactions within the bead wall itself that may increaseporosity.

A light stimulus may also be used to disrupt the beads. Numerous lighttriggers are possible and may include systems that use various moleculessuch as nanoparticles and chromophores capable of absorbing photons ofspecific ranges of wavelengths. For example, metal oxide coatings can beused as capsule triggers. UV irradiation of polyelectrolyte capsulescoated with SiO₂ may result in disintegration of the bead wall. In yetanother example, photo switchable materials such as azobenzene groupsmay be incorporated in the bead wall. Upon the application of UV orvisible light, chemicals such as these undergo a reversible cis-to-transisomerization upon absorption of photons. In this aspect, incorporationof photon switches result in a bead wall that may disintegrate or becomemore porous upon the application of a light trigger.

For example, in a non-limiting example of barcoding (e.g., stochasticbarcoding) illustrated in FIG. 2, after introducing cells such as singlecells onto a plurality of microwells of a microwell array at block 208,beads can be introduced onto the plurality of microwells of themicrowell array at block 212. Each microwell can comprise one bead. Thebeads can comprise a plurality of barcodes. A barcode can comprise a 5′amine region attached to a bead. The barcode can comprise a universallabel, a barcode sequence (e.g., a molecular label), a target-bindingregion, or any combination thereof.

The barcodes disclosed herein can be associated with (e.g., attached to)a solid support (e.g., a bead). The barcodes associated with a solidsupport can each comprise a barcode sequence selected from a groupcomprising at least 100 or 1000 barcode sequences with unique sequences.In some embodiments, different barcodes associated with a solid supportcan comprise barcode with different sequences. In some embodiments, apercentage of barcodes associated with a solid support comprises thesame cell label. For example, the percentage can be, or be about 60%,70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or a range betweenany two of these values. As another example, the percentage can be atleast, or be at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. Insome embodiments, barcodes associated with a solid support can have thesame cell label. The barcodes associated with different solid supportscan have different cell labels selected from a group comprising at least100 or 1000 cell labels with unique sequences.

The barcodes disclosed herein can be associated to (e.g., attached to) asolid support (e.g., a bead). In some embodiments, barcoding theplurality of targets in the sample can be performed with a solid supportincluding a plurality of synthetic particles associated with theplurality of barcodes. In some embodiments, the solid support caninclude a plurality of synthetic particles associated with the pluralityof barcodes. The spatial labels of the plurality of barcodes ondifferent solid supports can differ by at least one nucleotide. Thesolid support can, for example, include the plurality of barcodes in twodimensions or three dimensions. The synthetic particles can be beads.The beads can be silica gel beads, controlled pore glass beads, magneticbeads, Dynabeads, Sephadex/Sepharose beads, cellulose beads, polystyrenebeads, or any combination thereof. The solid support can include apolymer, a matrix, a hydrogel, a needle array device, an antibody, orany combination thereof. In some embodiments, the solid supports can befree floating. In some embodiments, the solid supports can be embeddedin a semi-solid or solid array. The barcodes may not be associated withsolid supports. The barcodes can be individual nucleotides. The barcodescan be associated with a substrate.

As used herein, the terms “tethered,” “attached,” and “immobilized,” areused interchangeably, and can refer to covalent or non-covalent meansfor attaching barcodes to a solid support. Any of a variety of differentsolid supports can be used as solid supports for attachingpre-synthesized barcodes or for in situ solid-phase synthesis ofbarcode.

In some embodiments, the solid support is a bead. The bead can compriseone or more types of solid, porous, or hollow sphere, ball, bearing,cylinder, or other similar configuration which a nucleic acid can beimmobilized (e.g., covalently or non-covalently). The bead can be, forexample, composed of plastic, ceramic, metal, polymeric material, or anycombination thereof. A bead can be, or comprise, a discrete particlethat is spherical (e.g., microspheres) or have a non-spherical orirregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical,oblong, or disc-shaped, and the like. In some embodiments, a bead can benon-spherical in shape.

Beads can comprise a variety of materials including, but not limited to,paramagnetic materials (e.g., magnesium, molybdenum, lithium, andtantalum), superparamagnetic materials (e.g., ferrite (Fe₃O₄; magnetite)nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt,some alloys thereof, and some rare earth metal compounds), ceramic,plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers,titanium, latex, Sepharose, agarose, hydrogel, polymer, cellulose,nylon, or any combination thereof.

In some embodiments, the bead (e.g., the bead to which the labels areattached) is a hydrogel bead. In some embodiments, the bead compriseshydrogel.

Some embodiments disclosed herein include one or more particles (forexample, beads). Each of the particles can comprise a plurality ofoligonucleotides (e.g., barcodes). Each of the plurality ofoligonucleotides can comprise a barcode sequence (e.g., a molecularlabel sequence), a cell label, and a target-binding region (e.g., anoligo(dT) sequence, a gene-specific sequence, a random multimer, or acombination thereof). The cell label sequence of each of the pluralityof oligonucleotides can be the same. The cell label sequences ofoligonucleotides on different particles can be different such that theoligonucleotides on different particles can be identified. The number ofdifferent cell label sequences can be different in differentimplementations. In some embodiments, the number of cell label sequencescan be, or be about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸,10⁹, a number or a range between any two of these values, or more. Insome embodiments, the number of cell label sequences can be at least, orbe at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸, or 10⁹. Insome embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, or more of the plurality of the particles include oligonucleotideswith the same cell sequence. In some embodiment, the plurality ofparticles that include oligonucleotides with the same cell sequence canbe at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, none ofthe plurality of the particles has the same cell label sequence.

The plurality of oligonucleotides on each particle can comprisedifferent barcode sequences (e.g., molecular labels). In someembodiments, the number of barcode sequences can be, or be about 10,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000,70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸, 10⁹, or a number or a rangebetween any two of these values. In some embodiments, the number ofbarcode sequences can be at least, or be at most 10, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,100000, 10⁶, 10⁷, 10⁸, or 10⁹. For example, at least 100 of theplurality of oligonucleotides comprise different barcode sequences. Asanother example, in a single particle, at least 100, 500, 1000, 5000,10000, 15000, 20000, 50000, a number or a range between any two of thesevalues, or more of the plurality of oligonucleotides comprise differentbarcode sequences. Some embodiments provide a plurality of the particlescomprising barcodes. In some embodiments, the ratio of an occurrence (ora copy or a number) of a target to be labeled and the different barcodesequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30,1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, eachof the plurality of oligonucleotides further comprises a sample label, auniversal label, or both. The particle can be, for example, ananoparticle or microparticle.

The size of the beads can vary. For example, the diameter of the beadcan range from 0.1 micrometer to 50 micrometer. In some embodiments, thediameter of the bead can be, or be about, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50 micrometer, or a number or a range between anytwo of these values.

The diameter of the bead can be related to the diameter of the wells ofthe substrate. In some embodiments, the diameter of the bead can be, orbe about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a numberor a range between any two of these values, longer or shorter than thediameter of the well. The diameter of the beads can be related to thediameter of a cell (e.g., a single cell entrapped by a well of thesubstrate). In some embodiments, the diameter of the bead can be atleast, or be at most, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% longer or shorter than the diameter of the well. The diameter ofthe beads can be related to the diameter of a cell (e.g., a single cellentrapped by a well of the substrate). In some embodiments, the diameterof the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 150%, 200%, 250%, 300%, or a number or a range between anytwo of these values, longer or shorter than the diameter of the cell. Insome embodiments, the diameter of the beads can be at least, or be atmost, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,250%, or 300% longer or shorter than the diameter of the cell.

A bead can be attached to and/or embedded in a substrate. A bead can beattached to and/or embedded in a gel, hydrogel, polymer and/or matrix.The spatial position of a bead within a substrate (e.g., gel, matrix,scaffold, or polymer) can be identified using the spatial label presenton the barcode on the bead which can serve as a location address.

Examples of beads can include, but are not limited to, streptavidinbeads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads,antibody conjugated beads (e.g., anti-immunoglobulin microbeads),protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbeads,anti-fluorochrome microbeads, and BcMag™ Carboxyl-Terminated MagneticBeads.

A bead can be associated with (e.g., impregnated with) quantum dots orfluorescent dyes to make it fluorescent in one fluorescence opticalchannel or multiple optical channels. A bead can be associated with ironoxide or chromium oxide to make it paramagnetic or ferromagnetic. Beadscan be identifiable. For example, a bead can be imaged using a camera. Abead can have a detectable code associated with the bead. For example, abead can comprise a barcode. A bead can change size, for example, due toswelling in an organic or inorganic solution. A bead can be hydrophobic.A bead can be hydrophilic. A bead can be biocompatible.

A solid support (e.g., a bead) can be visualized. The solid support cancomprise a visualizing tag (e.g., fluorescent dye). A solid support(e.g., a bead) can be etched with an identifier (e.g., a number). Theidentifier can be visualized through imaging the beads.

A solid support can comprise an insoluble, semi-soluble, or insolublematerial. A solid support can be referred to as “functionalized” when itincludes a linker, a scaffold, a building block, or other reactivemoiety attached thereto, whereas a solid support may be“nonfunctionalized” when it lack such a reactive moiety attachedthereto. The solid support can be employed free in solution, such as ina microtiter well format; in a flow-through format, such as in a column;or in a dipstick.

The solid support can comprise a membrane, paper, plastic, coatedsurface, flat surface, glass, slide, chip, or any combination thereof. Asolid support can take the form of resins, gels, microspheres, or othergeometric configurations. A solid support can comprise silica chips,microparticles, nanoparticles, plates, arrays, capillaries, flatsupports such as glass fiber filters, glass surfaces, metal surfaces(steel, gold silver, aluminum, silicon and copper), glass supports,plastic supports, silicon supports, chips, filters, membranes, microwellplates, slides, plastic materials including multiwell plates ormembranes (e.g., formed of polyethylene, polypropylene, polyamide,polyvinylidenedifluoride), and/or wafers, combs, pins or needles (e.g.,arrays of pins suitable for combinatorial synthesis or analysis) orbeads in an array of pits or nanoliter wells of flat surfaces such aswafers (e.g., silicon wafers), wafers with pits with or without filterbottoms.

The solid support can comprise a polymer matrix (e.g., gel, hydrogel).The polymer matrix may be able to permeate intracellular space (e.g.,around organelles). The polymer matrix may able to be pumped throughoutthe circulatory system.

Substrates and Microwell Array

As used herein, a substrate can refer to a type of solid support. Asubstrate can refer to a solid support that can comprise barcodes orstochastic barcodes of the disclosure. A substrate can, for example,comprise a plurality of microwells. For example, a substrate can be awell array comprising two or more microwells. In some embodiments, amicrowell can comprise a small reaction chamber of defined volume. Insome embodiments, a microwell can entrap one or more cells. In someembodiments, a microwell can entrap only one cell. In some embodiments,a microwell can entrap one or more solid supports. In some embodiments,a microwell can entrap only one solid support. In some embodiments, amicrowell entraps a single cell and a single solid support (e.g., abead). A microwell can comprise barcode reagents of the disclosure.

Methods of Barcoding

The disclosure provides for methods for estimating the number ofdistinct targets at distinct locations in a physical sample (e.g.,tissue, organ, tumor, cell). The methods can comprise placing barcodes(e.g., stochastic barcodes) in close proximity with the sample, lysingthe sample, associating distinct targets with the barcodes, amplifyingthe targets and/or digitally counting the targets. The method canfurther comprise analyzing and/or visualizing the information obtainedfrom the spatial labels on the barcodes. In some embodiments, a methodcomprises visualizing the plurality of targets in the sample. Mappingthe plurality of targets onto the map of the sample can includegenerating a two dimensional map or a three dimensional map of thesample. The two dimensional map and the three dimensional map can begenerated prior to or after barcoding (e.g., stochastically barcoding)the plurality of targets in the sample. Visualizing the plurality oftargets in the sample can include mapping the plurality of targets ontoa map of the sample. Mapping the plurality of targets onto the map ofthe sample can include generating a two dimensional map or a threedimensional map of the sample. The two dimensional map and the threedimensional map can be generated prior to or after barcoding theplurality of targets in the sample. In some embodiments, the twodimensional map and the three dimensional map can be generated before orafter lysing the sample. Lysing the sample before or after generatingthe two dimensional map or the three dimensional map can include heatingthe sample, contacting the sample with a detergent, changing the pH ofthe sample, or any combination thereof.

In some embodiments, barcoding the plurality of targets compriseshybridizing a plurality of barcodes with a plurality of targets tocreate barcoded targets (e.g., stochastically barcoded targets).Barcoding the plurality of targets can comprise generating an indexedlibrary of the barcoded targets. Generating an indexed library of thebarcoded targets can be performed with a solid support comprising theplurality of barcodes (e.g., stochastic barcodes).

Contacting a Sample and a Barcode

The disclosure provides for methods for contacting a sample (e.g.,cells) to a substrate of the disclosure. A sample comprising, forexample, a cell, organ, or tissue thin section, can be contacted tobarcodes (e.g., stochastic barcodes). The cells can be contacted, forexample, by gravity flow wherein the cells can settle and create amonolayer. The sample can be a tissue thin section. The thin section canbe placed on the substrate. The sample can be one-dimensional (e.g.,forms a planar surface). The sample (e.g., cells) can be spread acrossthe substrate, for example, by growing/culturing the cells on thesubstrate.

When barcodes are in close proximity to targets, the targets canhybridize to the barcode. The barcodes can be contacted at anon-depletable ratio such that each distinct target can associate with adistinct barcode of the disclosure. To ensure efficient associationbetween the target and the barcode, the targets can be cross-linked tobarcode.

Cell Lysis

Following the distribution of cells and barcodes, the cells can be lysedto liberate the target molecules. Cell lysis can be accomplished by anyof a variety of means, for example, by chemical or biochemical means, byosmotic shock, or by means of thermal lysis, mechanical lysis, oroptical lysis. Cells can be lysed by addition of a cell lysis buffercomprising a detergent (e.g., SDS, Li dodecyl sulfate, Triton X-100,Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), ordigestive enzymes (e.g., proteinase K, pepsin, or trypsin), or anycombination thereof. To increase the association of a target and abarcode, the rate of the diffusion of the target molecules can bealtered by for example, reducing the temperature and/or increasing theviscosity of the lysate.

In some embodiments, the sample can be lysed using a filter paper. Thefilter paper can be soaked with a lysis buffer on top of the filterpaper. The filter paper can be applied to the sample with pressure whichcan facilitate lysis of the sample and hybridization of the targets ofthe sample to the substrate.

In some embodiments, lysis can be performed by mechanical lysis, heatlysis, optical lysis, and/or chemical lysis. Chemical lysis can includethe use of digestive enzymes such as proteinase K, pepsin, and trypsin.Lysis can be performed by the addition of a lysis buffer to thesubstrate. A lysis buffer can comprise Tris HCl. A lysis buffer cancomprise at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris HCl. Alysis buffer can comprise at most about 0.01, 0.05, 0.1, 0.5, or 1 M ormore Tris HCL. A lysis buffer can comprise about 0.1 M Tris HCl. The pHof the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more. The pH of the lysis buffer can be at most about 1, 2, 3, 4, 5,6, 7, 8, 9,10, or more. In some embodiments, the pH of the lysis bufferis about 7.5. The lysis buffer can comprise a salt (e.g., LiCl). Theconcentration of salt in the lysis buffer can be at least about 0.1,0.5, or 1 M or more. The concentration of salt in the lysis buffer canbe at most about 0.1, 0.5, or 1 M or more. In some embodiments, theconcentration of salt in the lysis buffer is about 0.5M. The lysisbuffer can comprise a detergent (e.g., SDS, Li dodecyl sulfate, tritonX, tween, NP-40). The concentration of the detergent in the lysis buffercan be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%,0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or more. The concentration ofthe detergent in the lysis buffer can be at most about 0.0001%, 0.0005%,0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%,or more. In some embodiments, the concentration of the detergent in thelysis buffer is about 1% Li dodecyl sulfate. The time used in the methodfor lysis can be dependent on the amount of detergent used. In someembodiments, the more detergent used, the less time needed for lysis.The lysis buffer can comprise a chelating agent (e.g., EDTA and EGTA).The concentration of a chelating agent in the lysis buffer can be atleast about 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration ofa chelating agent in the lysis buffer can be at most about 1, 5, 10, 15,20, 25, or 30 mM or more. In some embodiments, the concentration ofchelating agent in the lysis buffer is about 10 mM. The lysis buffer cancomprise a reducing reagent (e.g., beta-mercaptoethanol, DTT). Theconcentration of the reducing reagent in the lysis buffer can be atleast about 1, 5, 10, 15, or 20 mM or more. The concentration of thereducing reagent in the lysis buffer can be at most about 1, 5, 10, 15,or 20 mM or more. In some embodiments, the concentration of reducingreagent in the lysis buffer is about 5 mM. In some embodiments, a lysisbuffer can comprise about 0.1M TrisHCl, about pH 7.5, about 0.5M LiCl,about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or30° C. Lysis can be performed for about 1, 5, 10, 15, or 20 or moreminutes. A lysed cell can comprise at least about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules. A lysed cell can comprise at most about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules.

Attachment of Barcodes to Target Nucleic Acid Molecules

Following lysis of the cells and release of nucleic acid moleculestherefrom, the nucleic acid molecules can randomly associate with thebarcodes of the co-localized solid support. Association can comprisehybridization of a barcode's target recognition region to acomplementary portion of the target nucleic acid molecule (e.g.,oligo(dT) of the barcode can interact with a poly(A) tail of a target).The assay conditions used for hybridization (e.g., buffer pH, ionicstrength, temperature, etc.) can be chosen to promote formation ofspecific, stable hybrids. In some embodiments, the nucleic acidmolecules released from the lysed cells can associate with the pluralityof probes on the substrate (e.g., hybridize with the probes on thesubstrate). When the probes comprise oligo(dT), mRNA molecules canhybridize to the probes and be reverse transcribed. The oligo(dT)portion of the oligonucleotide can act as a primer for first strandsynthesis of the cDNA molecule. For example, in a non-limiting exampleof barcoding illustrated in FIG. 2, at block 216, mRNA molecules canhybridize to barcodes on beads. For example, single-stranded nucleotidefragments can hybridize to the target-binding regions of barcodes.

Attachment can further comprise ligation of a barcode's targetrecognition region and a portion of the target nucleic acid molecule.For example, the target binding region can comprise a nucleic acidsequence that can be capable of specific hybridization to a restrictionsite overhang (e.g., an EcoRI sticky-end overhang). The assay procedurecan further comprise treating the target nucleic acids with arestriction enzyme (e.g., EcoRI) to create a restriction site overhang.The barcode can then be ligated to any nucleic acid molecule comprisinga sequence complementary to the restriction site overhang. A ligase(e.g., T4 DNA ligase) can be used to join the two fragments.

For example, in a non-limiting example of barcoding illustrated in FIG.2, at block 220, the labeled targets from a plurality of cells (or aplurality of samples) (e.g., target-barcode molecules) can besubsequently pooled, for example, into a tube. The labeled targets canbe pooled by, for example, retrieving the barcodes and/or the beads towhich the target-barcode molecules are attached.

The retrieval of solid support-based collections of attachedtarget-barcode molecules can be implemented by use of magnetic beads andan externally-applied magnetic field. Once the target-barcode moleculeshave been pooled, all further processing can proceed in a singlereaction vessel. Further processing can include, for example, reversetranscription reactions, amplification reactions, cleavage reactions,dissociation reactions, and/or nucleic acid extension reactions. Furtherprocessing reactions can be performed within the microwells, that is,without first pooling the labeled target nucleic acid molecules from aplurality of cells.

Reverse Transcription or Nucleic Acid Extension

The disclosure provides for a method to create a target-barcodeconjugate using reverse transcription (e.g., at block 224 of FIG. 2) ornucleic acid extension. The target-barcode conjugate can comprise thebarcode and a complementary sequence of all or a portion of the targetnucleic acid (i.e., a barcoded cDNA molecule, such as a stochasticallybarcoded cDNA molecule). Reverse transcription of the associated RNAmolecule can occur by the addition of a reverse transcription primeralong with the reverse transcriptase. The reverse transcription primercan be an oligo(dT) primer, a random hexanucleotide primer, or atarget-specific oligonucleotide primer. Oligo(dT) primers can be, or canbe about, 12-18 nucleotides in length and bind to the endogenous poly(A)tail at the 3′ end of mammalian mRNA. Random hexanucleotide primers canbind to mRNA at a variety of complementary sites. Target-specificoligonucleotide primers typically selectively prime the mRNA ofinterest.

In some embodiments, reverse transcription of an mRNA molecule to alabeled-RNA molecule can occur by the addition of a reversetranscription primer. In some embodiments, the reverse transcriptionprimer is an oligo(dT) primer, random hexanucleotide primer, or atarget-specific oligonucleotide primer. Generally, oligo(dT) primers are12-18 nucleotides in length and bind to the endogenous poly(A) tail atthe 3′ end of mammalian mRNA. Random hexanucleotide primers can bind tomRNA at a variety of complementary sites. Target-specificoligonucleotide primers typically selectively prime the mRNA ofinterest.

In some embodiments, a target is a cDNA molecule. For example, an mRNAmolecule can be reverse transcribed using a reverse transcriptase, suchas Moloney murine leukemia virus (MMLV) reverse transcriptase, togenerate a cDNA molecule with a poly(dC) tail. A barcode can include atarget binding region with a poly(dG) tail. Upon base pairing betweenthe poly(dG) tail of the barcode and the poly(dC) tail of the cDNAmolecule, the reverse transcriptase switches template strands, fromcellular RNA molecule to the barcode, and continues replication to the5′ end of the barcode. By doing so, the resulting cDNA molecule containsthe sequence of the barcode (such as the molecular label) on the 3′ endof the cDNA molecule.

Reverse transcription can occur repeatedly to produce multiplelabeled-cDNA molecules. The methods disclosed herein can compriseconducting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methodcan comprise conducting at least about 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

Amplification

One or more nucleic acid amplification reactions (e.g., at block 228 ofFIG. 2) can be performed to create multiple copies of the labeled targetnucleic acid molecules. Amplification can be performed in a multiplexedmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. The amplification reaction can be used to add sequencingadaptors to the nucleic acid molecules. The amplification reactions cancomprise amplifying at least a portion of a sample label, if present.The amplification reactions can comprise amplifying at least a portionof the cellular label and/or barcode sequence (e.g., a molecular label).The amplification reactions can comprise amplifying at least a portionof a sample tag, a cell label, a spatial label, a barcode sequence(e.g., a molecular label), a target nucleic acid, or a combinationthereof. The amplification reactions can comprise amplifying 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a rangeor a number between any two of these values, of the plurality of nucleicacids. The method can further comprise conducting one or more cDNAsynthesis reactions to produce one or more cDNA copies of target-barcodemolecules comprising a sample label, a cell label, a spatial label,and/or a barcode sequence (e.g., a molecular label).

In some embodiments, amplification can be performed using a polymerasechain reaction (PCR). As used herein, PCR can refer to a reaction forthe in vitro amplification of specific DNA sequences by the simultaneousprimer extension of complementary strands of DNA. As used herein, PCRcan encompass derivative forms of the reaction, including but notlimited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,multiplexed PCR, digital PCR, and assembly PCR.

Amplification of the labeled nucleic acids can comprise non-PCR basedmethods. Examples of non-PCR based methods include, but are not limitedto, multiple displacement amplification (MDA), transcription-mediatedamplification (TMA), nucleic acid sequence-based amplification (NASBA),strand displacement amplification (SDA), real-time SDA, rolling circleamplification, or circle-to-circle amplification. Other non-PCR-basedamplification methods include multiple cycles of DNA-dependent RNApolymerase-driven RNA transcription amplification or RNA-directed DNAsynthesis and transcription to amplify DNA or RNA targets, a ligasechain reaction (LCR), and a Qβ replicase (Qβ) method, use of palindromicprobes, strand displacement amplification, oligonucleotide-drivenamplification using a restriction endonuclease, an amplification methodin which a primer is hybridized to a nucleic acid sequence and theresulting duplex is cleaved prior to the extension reaction andamplification, strand displacement amplification using a nucleic acidpolymerase lacking 5′ exonuclease activity, rolling circleamplification, and ramification extension amplification (RAM). In someembodiments, the amplification does not produce circularizedtranscripts.

In some embodiments, the methods disclosed herein further compriseconducting a polymerase chain reaction on the labeled nucleic acid(e.g., labeled-RNA, labeled-DNA, labeled-cDNA) to produce a labeledamplicon (e.g., a stochastically labeled amplicon). The labeled ampliconcan be double-stranded molecule. The double-stranded molecule cancomprise a double-stranded RNA molecule, a double-stranded DNA molecule,or a RNA molecule hybridized to a DNA molecule. One or both of thestrands of the double-stranded molecule can comprise a sample label, aspatial label, a cell label, and/or a barcode sequence (e.g., amolecular label). The labeled amplicon can be a single-strandedmolecule. The single-stranded molecule can comprise DNA, RNA, or acombination thereof. The nucleic acids of the disclosure can comprisesynthetic or altered nucleic acids.

Amplification can comprise use of one or more non-natural nucleotides.Non-natural nucleotides can comprise photolabile or triggerablenucleotides. Examples of non-natural nucleotides can include, but arenot limited to, peptide nucleic acid (PNA), morpholino and lockednucleic acid (LNA), as well as glycol nucleic acid (GNA) and threosenucleic acid (TNA). Non-natural nucleotides can be added to one or morecycles of an amplification reaction. The addition of the non-naturalnucleotides can be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions can comprise the useof one or more primers. The one or more primers can comprise, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or morenucleotides. The one or more primers can comprise at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one ormore primers can comprise less than 12-15 nucleotides. The one or moreprimers can anneal to at least a portion of the plurality of labeledtargets (e.g., stochastically labeled targets). The one or more primerscan anneal to the 3′ end or 5′ end of the plurality of labeled targets.The one or more primers can anneal to an internal region of theplurality of labeled targets. The internal region can be at least about50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3′ endsthe plurality of labeled targets. The one or more primers can comprise afixed panel of primers. The one or more primers can comprise at leastone or more custom primers. The one or more primers can comprise atleast one or more control primers. The one or more primers can compriseat least one or more gene-specific primers.

The one or more primers can comprise a universal primer. The universalprimer can anneal to a universal primer binding site. The one or morecustom primers can anneal to a first sample label, a second samplelabel, a spatial label, a cell label, a barcode sequence (e.g., amolecular label), a target, or any combination thereof. The one or moreprimers can comprise a universal primer and a custom primer. The customprimer can be designed to amplify one or more targets. The targets cancomprise a subset of the total nucleic acids in one or more samples. Thetargets can comprise a subset of the total labeled targets in one ormore samples. The one or more primers can comprise at least 96 or morecustom primers. The one or more primers can comprise at least 960 ormore custom primers. The one or more primers can comprise at least 9600or more custom primers. The one or more custom primers can anneal to twoor more different labeled nucleic acids. The two or more differentlabeled nucleic acids can correspond to one or more genes.

Any amplification scheme can be used in the methods of the presentdisclosure. For example, in one scheme, the first round PCR can amplifymolecules attached to the bead using a gene specific primer and a primeragainst the universal Illumina sequencing primer 1 sequence. The secondround of PCR can amplify the first PCR products using a nested genespecific primer flanked by Illumina sequencing primer 2 sequence, and aprimer against the universal Illumina sequencing primer 1 sequence. Thethird round of PCR adds P5 and P7 and sample index to turn PCR productsinto an Illumina sequencing library. Sequencing using 150 bp×2sequencing can reveal the cell label and barcode sequence (e.g.,molecular label) on read 1, the gene on read 2, and the sample index onindex 1 read.

In some embodiments, nucleic acids can be removed from the substrateusing chemical cleavage. For example, a chemical group or a modifiedbase present in a nucleic acid can be used to facilitate its removalfrom a solid support. For example, an enzyme can be used to remove anucleic acid from a substrate. For example, a nucleic acid can beremoved from a substrate through a restriction endonuclease digestion.For example, treatment of a nucleic acid containing a dUTP or ddUTP withuracil-d-glycosylase (UDG) can be used to remove a nucleic acid from asubstrate. For example, a nucleic acid can be removed from a substrateusing an enzyme that performs nucleotide excision, such as a baseexcision repair enzyme, such as an apurinic/apyrimidinic (AP)endonuclease. In some embodiments, a nucleic acid can be removed from asubstrate using a photocleavable group and light. In some embodiments, acleavable linker can be used to remove a nucleic acid from thesubstrate. For example, the cleavable linker can comprise at least oneof biotin/avidin, biotin/streptavidin, biotin/neutravidin, Ig-protein A,a photo-labile linker, acid or base labile linker group, or an aptamer.

When the probes are gene-specific, the molecules can hybridize to theprobes and be reverse transcribed and/or amplified. In some embodiments,after the nucleic acid has been synthesized (e.g., reverse transcribed),it can be amplified. Amplification can be performed in a multiplexmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. Amplification can add sequencing adaptors to the nucleicacid.

In some embodiments, amplification can be performed on the substrate,for example, with bridge amplification. cDNAs can be homopolymer tailedin order to generate a compatible end for bridge amplification usingoligo(dT) probes on the substrate. In bridge amplification, the primerthat is complementary to the 3′ end of the template nucleic acid can bethe first primer of each pair that is covalently attached to the solidparticle. When a sample containing the template nucleic acid iscontacted with the particle and a single thermal cycle is performed, thetemplate molecule can be annealed to the first primer and the firstprimer is elongated in the forward direction by addition of nucleotidesto form a duplex molecule consisting of the template molecule and anewly formed DNA strand that is complementary to the template. In theheating step of the next cycle, the duplex molecule can be denatured,releasing the template molecule from the particle and leaving thecomplementary DNA strand attached to the particle through the firstprimer. In the annealing stage of the annealing and elongation step thatfollows, the complementary strand can hybridize to the second primer,which is complementary to a segment of the complementary strand at alocation removed from the first primer. This hybridization can cause thecomplementary strand to form a bridge between the first and secondprimers secured to the first primer by a covalent bond and to the secondprimer by hybridization. In the elongation stage, the second primer canbe elongated in the reverse direction by the addition of nucleotides inthe same reaction mixture, thereby converting the bridge to adouble-stranded bridge. The next cycle then begins, and thedouble-stranded bridge can be denatured to yield two single-strandednucleic acid molecules, each having one end attached to the particlesurface via the first and second primers, respectively, with the otherend of each unattached. In the annealing and elongation step of thissecond cycle, each strand can hybridize to a further complementaryprimer, previously unused, on the same particle, to form newsingle-strand bridges. The two previously unused primers that are nowhybridized elongate to convert the two new bridges to double-strandbridges.

The amplification reactions can comprise amplifying at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of theplurality of nucleic acids.

Amplification of the labeled nucleic acids can comprise PCR-basedmethods or non-PCR based methods. Amplification of the labeled nucleicacids can comprise exponential amplification of the labeled nucleicacids. Amplification of the labeled nucleic acids can comprise linearamplification of the labeled nucleic acids. Amplification can beperformed by polymerase chain reaction (PCR). PCR can refer to areaction for the in vitro amplification of specific DNA sequences by thesimultaneous primer extension of complementary strands of DNA. PCR canencompass derivative forms of the reaction, including but not limitedto, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexedPCR, digital PCR, suppression PCR, semi-suppressive PCR and assemblyPCR.

In some embodiments, amplification of the labeled nucleic acidscomprises non-PCR based methods. Examples of non-PCR based methodsinclude, but are not limited to, multiple displacement amplification(MDA), transcription-mediated amplification (TMA), nucleic acidsequence-based amplification (NASBA), strand displacement amplification(SDA), real-time SDA, rolling circle amplification, or circle-to-circleamplification. Other non-PCR-based amplification methods includemultiple cycles of DNA-dependent RNA polymerase-driven RNA transcriptionamplification or RNA-directed DNA synthesis and transcription to amplifyDNA or RNA targets, a ligase chain reaction (LCR), a Qβ replicase (Qβ),use of palindromic probes, strand displacement amplification,oligonucleotide-driven amplification using a restriction endonuclease,an amplification method in which a primer is hybridized to a nucleicacid sequence and the resulting duplex is cleaved prior to the extensionreaction and amplification, strand displacement amplification using anucleic acid polymerase lacking 5′ exonuclease activity, rolling circleamplification, and/or ramification extension amplification (RAM).

In some embodiments, the methods disclosed herein further compriseconducting a nested polymerase chain reaction on the amplified amplicon(e.g., target). The amplicon can be double-stranded molecule. Thedouble-stranded molecule can comprise a double-stranded RNA molecule, adouble-stranded DNA molecule, or a RNA molecule hybridized to a DNAmolecule. One or both of the strands of the double-stranded molecule cancomprise a sample tag or molecular identifier label. Alternatively, theamplicon can be a single-stranded molecule. The single-stranded moleculecan comprise DNA, RNA, or a combination thereof. The nucleic acids ofthe present invention can comprise synthetic or altered nucleic acids.

In some embodiments, the method comprises repeatedly amplifying thelabeled nucleic acid to produce multiple amplicons. The methodsdisclosed herein can comprise conducting at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplificationreactions. Alternatively, the method comprises conducting at least about25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100amplification reactions.

Amplification can further comprise adding one or more control nucleicacids to one or more samples comprising a plurality of nucleic acids.Amplification can further comprise adding one or more control nucleicacids to a plurality of nucleic acids. The control nucleic acids cancomprise a control label.

Amplification can comprise use of one or more non-natural nucleotides.Non-natural nucleotides can comprise photolabile and/or triggerablenucleotides. Examples of non-natural nucleotides include, but are notlimited to, peptide nucleic acid (PNA), morpholino and locked nucleicacid (LNA), as well as glycol nucleic acid (GNA) and threose nucleicacid (TNA). Non-natural nucleotides can be added to one or more cyclesof an amplification reaction. The addition of the non-naturalnucleotides can be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions can comprise the useof one or more primers. The one or more primers can comprise one or moreoligonucleotides. The one or more oligonucleotides can comprise at leastabout 7-9 nucleotides. The one or more oligonucleotides can compriseless than 12-15 nucleotides. The one or more primers can anneal to atleast a portion of the plurality of labeled nucleic acids. The one ormore primers can anneal to the 3′ end and/or 5′ end of the plurality oflabeled nucleic acids. The one or more primers can anneal to an internalregion of the plurality of labeled nucleic acids. The internal regioncan be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000nucleotides from the 3′ ends the plurality of labeled nucleic acids. Theone or more primers can comprise a fixed panel of primers. The one ormore primers can comprise at least one or more custom primers. The oneor more primers can comprise at least one or more control primers. Theone or more primers can comprise at least one or more housekeeping geneprimers. The one or more primers can comprise a universal primer. Theuniversal primer can anneal to a universal primer binding site. The oneor more custom primers can anneal to the first sample tag, the secondsample tag, the molecular identifier label, the nucleic acid or aproduct thereof. The one or more primers can comprise a universal primerand a custom primer. The custom primer can be designed to amplify one ormore target nucleic acids. The target nucleic acids can comprise asubset of the total nucleic acids in one or more samples. In someembodiments, the primers are the probes attached to the array of thedisclosure.

In some embodiments, barcoding (e.g., stochastically barcoding) theplurality of targets in the sample further comprises generating anindexed library of the barcoded targets (e.g., stochastically barcodedtargets) or barcoded fragments of the targets. The barcode sequences ofdifferent barcodes (e.g., the molecular labels of different stochasticbarcodes) can be different from one another. Generating an indexedlibrary of the barcoded targets includes generating a plurality ofindexed polynucleotides from the plurality of targets in the sample. Forexample, for an indexed library of the barcoded targets comprising afirst indexed target and a second indexed target, the label region ofthe first indexed polynucleotide can differ from the label region of thesecond indexed polynucleotide by, by about, by at least, or by at most,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or a number or a rangebetween any two of these values, nucleotides. In some embodiments,generating an indexed library of the barcoded targets includescontacting a plurality of targets, for example mRNA molecules, with aplurality of oligonucleotides including a poly(T) region and a labelregion; and conducting a first strand synthesis using a reversetranscriptase to produce single-strand labeled cDNA molecules eachcomprising a cDNA region and a label region, wherein the plurality oftargets includes at least two mRNA molecules of different sequences andthe plurality of oligonucleotides includes at least two oligonucleotidesof different sequences. Generating an indexed library of the barcodedtargets can further comprise amplifying the single-strand labeled cDNAmolecules to produce double-strand labeled cDNA molecules; andconducting nested PCR on the double-strand labeled cDNA molecules toproduce labeled amplicons. In some embodiments, the method can includegenerating an adaptor-labeled amplicon.

Barcoding (e.g., stochastic barcoding) can include using nucleic acidbarcodes or tags to label individual nucleic acid (e.g., DNA or RNA)molecules. In some embodiments, it involves adding DNA barcodes or tagsto cDNA molecules as they are generated from mRNA. Nested PCR can beperformed to minimize PCR amplification bias. Adaptors can be added forsequencing using, for example, next generation sequencing (NGS). Thesequencing results can be used to determine cell labels, molecularlabels, and sequences of nucleotide fragments of the one or more copiesof the targets, for example at block 232 of FIG. 2.

FIG. 3 is a schematic illustration showing a non-limiting exemplaryprocess of generating an indexed library of the barcoded targets (e.g.,stochastically barcoded targets), such as barcoded mRNAs or fragmentsthereof. As shown in step 1, the reverse transcription process canencode each mRNA molecule with a unique molecular label sequence, a celllabel sequence, and a universal PCR site. In particular, RNA molecules302 can be reverse transcribed to produce labeled cDNA molecules 304,including a cDNA region 306, by hybridization (e.g., stochastichybridization) of a set of barcodes (e.g., stochastic barcodes) 310 tothe poly(A) tail region 308 of the RNA molecules 302. Each of thebarcodes 310 can comprise a target-binding region, for example apoly(dT) region 312, a label region 314 (e.g., a barcode sequence or amolecule), and a universal PCR region 316.

In some embodiments, the cell label sequence can include 3 to 20nucleotides. In some embodiments, the molecular label sequence caninclude 3 to 20 nucleotides. In some embodiments, each of the pluralityof stochastic barcodes further comprises one or more of a universallabel and a cell label, wherein universal labels are the same for theplurality of stochastic barcodes on the solid support and cell labelsare the same for the plurality of stochastic barcodes on the solidsupport. In some embodiments, the universal label can include 3 to 20nucleotides. In some embodiments, the cell label comprises 3 to 20nucleotides.

In some embodiments, the label region 314 can include a barcode sequenceor a molecular label 318 and a cell label 320. In some embodiments, thelabel region 314 can include one or more of a universal label, adimension label, and a cell label. The barcode sequence or molecularlabel 318 can be, can be about, can be at least, or can be at most, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or anumber or a range between any of these values, of nucleotides in length.The cell label 320 can be, can be about, can be at least, or can be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any of these values, of nucleotidesin length. The universal label can be, can be about, can be at least, orcan be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any of these values, ofnucleotides in length. Universal labels can be the same for theplurality of stochastic barcodes on the solid support and cell labelsare the same for the plurality of stochastic barcodes on the solidsupport. The dimension label can be, can be about, can be at least, orcan be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any of these values, ofnucleotides in length.

In some embodiments, the label region 314 can comprise, comprise about,comprise at least, or comprise at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, or a number or a range between any of these values, differentlabels, such as a barcode sequence or a molecular label 318 and a celllabel 320. Each label can be, can be about, can be at least, or can beat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any of these values, of nucleotidesin length. A set of barcodes or stochastic barcodes 310 can contain,contain about, contain at least, or can be at most, 10, 20, 40, 50, 70,80, 90, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10²⁰, or a number or a range between any of these values,barcodes or stochastic barcodes 310. And the set of barcodes orstochastic barcodes 310 can, for example, each contain a unique labelregion 314. The labeled cDNA molecules 304 can be purified to removeexcess barcodes or stochastic barcodes 310. Purification can compriseAmpure bead purification.

As shown in step 2, products from the reverse transcription process instep 1 can be pooled into 1 tube and PCR amplified with a 1^(st) PCRprimer pool and a 1^(st) universal PCR primer. Pooling is possiblebecause of the unique label region 314. In particular, the labeled cDNAmolecules 304 can be amplified to produce nested PCR labeled amplicons322. Amplification can comprise multiplex PCR amplification.Amplification can comprise a multiplex PCR amplification with 96multiplex primers in a single reaction volume. In some embodiments,multiplex PCR amplification can utilize, utilize about, utilize atleast, or utilize at most, 10, 20, 40, 50, 70, 80, 90, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10²⁰, or anumber or a range between any of these values, multiplex primers in asingle reaction volume. Amplification can comprise using a 1^(st) PCRprimer pool 324 comprising custom primers 326A-C targeting specificgenes and a universal primer 328. The custom primers 326 can hybridizeto a region within the cDNA portion 306′ of the labeled cDNA molecule304. The universal primer 328 can hybridize to the universal PCR region316 of the labeled cDNA molecule 304.

As shown in step 3 of FIG. 3, products from PCR amplification in step 2can be amplified with a nested PCR primers pool and a 2^(nd) universalPCR primer. Nested PCR can minimize PCR amplification bias. Inparticular, the nested PCR labeled amplicons 322 can be furtheramplified by nested PCR. The nested PCR can comprise multiplex PCR withnested PCR primers pool 330 of nested PCR primers 332 a-c and a 2^(nd)universal PCR primer 328′ in a single reaction volume. The nested PCRprimer pool 328 can contain, contain about, contain at least, or containat most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any of these values, different nested PCR primers 330. Thenested PCR primers 332 can contain an adaptor 334 and hybridize to aregion within the cDNA portion 306″ of the labeled amplicon 322. Theuniversal primer 328′ can contain an adaptor 336 and hybridize to theuniversal PCR region 316 of the labeled amplicon 322. Thus, step 3produces adaptor-labeled amplicon 338. In some embodiments, nested PCRprimers 332 and the 2^(nd) universal PCR primer 328′ may not contain theadaptors 334 and 336. The adaptors 334 and 336 can instead be ligated tothe products of nested PCR to produce adaptor-labeled amplicon 338.

As shown in step 4, PCR products from step 3 can be PCR amplified forsequencing using library amplification primers. In particular, theadaptors 334 and 336 can be used to conduct one or more additionalassays on the adaptor-labeled amplicon 338. The adaptors 334 and 336 canbe hybridized to primers 340 and 342. The one or more primers 340 and342 can be PCR amplification primers. The one or more primers 340 and342 can be sequencing primers. The one or more adaptors 334 and 336 canbe used for further amplification of the adaptor-labeled amplicons 338.The one or more adaptors 334 and 336 can be used for sequencing theadaptor-labeled amplicon 338. The primer 342 can contain a plate index344 so that amplicons generated using the same set of barcodes orstochastic barcodes 310 can be sequenced in one sequencing reactionusing next generation sequencing (NGS).

Methods and Compositions for Single Cell Secretomics

There are provided, in some embodiments, systems, methods, compositions,and kits for single cell secretomics. The methods and compositionsdisclosed herein can determine the number of copies of one or moresecreted factors secreted by a single cell (e.g., secretomics, secretedfactor profiling). There are provided, in some embodiments, compositionsand methods for single cell secretome analysis. Some embodimentsdescribed herein employ antibodies capable of binding secreted factors,and said antibodies can be associated with oligonucleotides. In someembodiments, the methods and compositions provided herein are compatiblewith single cell analysis systems, workflows, and platforms (e.g., BDRhapsody). There is a need for compositions and methods for analyzingthe secretome profile of individual cells. Currently available methodsof secretome profiling (e.g., fluorescence- or microarray chip-based)are high-cost and low-throughput, and are less quantitative than thesecretome profiling methods and compositions provided herein. In someembodiments, secreted proteins of single cells are captured by syntheticbeads (a cell mimic) with multiple antibodies (specific for saidsecreted proteins) conjugated thereto. In some embodiments, said beadscan then be probed with secreted factor binding-reagents as describedherein. Said secreted factor binding-reagents can comprise a secretedfactor-binding reagent specific oligonucleotide comprising a uniquefactor identifier sequence for the secreted factor-binding reagent.Barcoding and sequence analysis of said secreted factor-binding reagentspecific oligonucleotides as described herein can enable high-throughputsingle cell secretome analysis.

There are provided, in some embodiments solid supports (e.g., beads,first solid supports, second solid supports) that are cell-sized. Saidsolid supports can comprise a functional surface for antibodyconjugation (e.g., conjugation of antibodies versus secreted proteins).The method can comprise incubation of single cells with saidantibody-coated solid support. The incubation can take place inpartitions (e.g., wells). Partition size can be optimized to capturemost of secreted proteins. The time, media, and/or temperature ofincubation can be varied depending on the needs of the user. Cells can,depending on the embodiment, die during the incubation; however, in somesuch embodiments, the secretome information is still valuable. Thus, thecompositions and methods provided herein can still acquire usefulsecretome information even in cases of cell death. The solid supportscan be collected from said partitions and stained with the secretedfactor-binding reagents provided herein. The staining can bemultiplexed. In some embodiments provided herein, flow Abs can be alsoadded for sorting to enrich certain secretomic types. In someembodiments, solid supports are stained with antibodies comprising adetectable moiety that enable the sorting of solid supports by flowcytometry for one or more desired properties. The method can comprisewashing of unbound the secreted factor-binding reagents. The solidsupports can be loaded into a plurality of partitions (e.g., wells,droplets, partitions of a single cell analysis platform such as BDRhapsody). A single partition of the plurality of partitions cancomprise a single solid support. A single partition of the plurality ofpartitions can comprise a plurality of oligonucleotide barcodes. Asingle partition of the plurality of partitions can comprise a barcodingparticle (e.g., a single third solid support as described herein).Barcoding of barcoded secreted factor-binding reagent specificoligonucleotides with said oligonucleotide barcodes can be performed asdescribed herein. Library generation and obtaining the sequenceinformation can yield secretome data.

There are provided, in some embodiments, compositions and methods forsimultaneous high-throughput single cell secretome and transcriptomeanalysis. In some such embodiments, the solid support (e.g. second solidsupport) comprises a plurality of capture probes and a plurality ofanchor probes. Each of the plurality of anchor probes can be capable ofspecifically binding to a surface cellular target to form single cellsassociated with a second solid support. Barcoding and sequence analysisof said secreted factor-binding reagent specific oligonucleotides aswell as the nucleic acid targets of the associated single cells asdescribed herein can enable high-throughput single cell secretome andtranscriptome analysis. In some embodiments, single cell secretomeanalysis and single cell protein profiling can be performed using thecompositions and methods provided herein. In some embodiments,compositions and methods for simultaneous single cell secretomeanalysis, transcriptome analysis, and protein profiling are described.Embodiments of using AbOs to determine protein expression profiles insingle cells and tracking sample origins have been described in U.S.patent application Ser. No. 15/715,028, published as U.S. PatentApplication Publication No. 2018/0088112, and U.S. patent applicationSer. No. 15/937,713; the content of each is incorporated by referenceherein in its entirety.

In some embodiments, the solid support (e.g., bead, first solid support,second solid support) comprising a plurality of capture probes (e.g.,multiple different antibodies for secreted protein) is a cell mimic. Asingle cell and one of said solid supports can located in a partition(e.g., well) of a plurality of partitions (e.g., a culture plate) withmedia so secreted proteins will be captured by capture probes on thesolid support from the given cell. Then the solid support can be stainedwith secreted factor binding reagents that can detect the capturedproteins and those solid supports can be loaded onto a plurality ofpartitions (e.g., wells, droplets, partitions of a single cell analysisplatform such as BD Rhapsody) for library generation. Said secretedfactor binding-reagents can comprise a secreted factor-binding reagentspecific oligonucleotide comprising a unique factor identifier sequencefor the secreted factor-binding reagent. The secreted factor-bindingreagent specific oligonucleotide library can be sequenced and generatethe secretome information of single cells. Currently available methodsfor detecting secreted proteins have a limitation on the number ofproteins that can be detected due to the number of fluorescence markersthat can used in the microscope or flow cytometry analysis. Thecompositions and methods provided herein remove this limitation and canenable detection of secreted proteins with as many pairs of captureprobe/secreted factor-binding reagent needed by the user. Moreover, asdescribed therein, the disclosed methods can comprise the capture ofboth secreted protein and single cells for the analysis of thetranscriptome and secretome of a single cell. Systems, methods,compositions, and kits for measuring secreted factors from cellsemploying (i) bispecific probes comprising anchor probe(s) capable ofspecifically binding to a surface cellular target of a cell and captureprobe(s) capable of specifically binding to a secreted factor secretedby a cell that is associated with the capture probe, and/or (ii)secreted factor-binding reagents capable of specifically binding to asecreted factor bound by a capture probe, are described in the U.S.Patent Application No. 62/962,927, filed Jan. 17, 2020, entitled“METHODS AND COMPOSITIONS FOR SINGLE CELL SECRETOMICS”, the content ofwhich is incorporated herein by reference in its entirety.

Currently available methods of single cell secretome profiling, such asthose using fluorescence detection on microarray chips, suffer from thedeficiencies of being less quantitative due to the fluorescenceintensity differences and low potential to capture transcriptome ofgiven cell. The limitations of fluorescence intensity difference-basedmethods and low number of secreted proteins that can be detected from agiven cell are solved with the methods and compositions provided herein,in some embodiments, by utilizing the sequence analysis of secretedfactor-binding reagent specific oligonucleotides, which are not limitedby fluorescent markers and are more quantitative due to the ability tocount the number of molecules (instead of fluorescent intensity).

Cytokines and other proteins released by the cell are of keen interestto immunologists and other cell biologists. Traditional methods fordetecting and measuring secreted proteins are typically measured in bulk(rather than at the single cell level). For example, currently availablemethods include bead-based assays and ELISA for studying secretedfactors in bulk. Therefore, single cell quantification and cellularphenotype analysis are missing in the data. As with the comparison offlow cytometry to traditional western blots, there is tremendous valuein studying the individual cells from a heterogenous mixture of cells.The methods and compositions provided herein enable detection andrelative quantification of secreted proteins of individual cells in aheterogeneous mixture. Oligonucleotide barcoded detection probes (e.g.,secreted factor-binding reagent specific oligonucleotides) can beoptimized for single cell genomics analysis. The secreted factoranalysis methods provided herein can be compatible with other analysestechniques for single cell multiomics platforms. Disclosed hereininclude methods and compositions enabling rapid adoption of single-cellsecretomic assays across a flexible portfolio of targets without theneed for specialized instrumentation. In some embodiments, the methodsdisclosed herein can provide the ability to assay secreted proteinswithout compromising cell-viability, and thus can enable the sorting oflive cells based on their protein secretion profile. Additionally, themethods provided herein enable a broader suite of single cell omic datadownstream of cell preparation.

The use an oligo-barcoded detection probe (e.g., an secreted factorbinding reagent) as provided herein enables, for the first time, theability to assess secreted factors from individual cells simultaneouslywith surface proteins (e.g., cellular component targets) andintracellular transcript (mRNA). The methods and compositions providedherein enable, for the first time, single cell secretion analysis onsingle cell genomic platforms.

In some embodiments of the methods and compositions provided herein, aDNA cellular component binding reagent specific oligonucleotide (e.g.,an antibody oligonucleotide) is hybridized to an oligonucleotide barcodeand extended to enable a separate, but parallel workflow for proteinquantitation and mRNA quantitation from the same beads, as described inU.S. application Ser. No. 17/147,272, the content of which isincorporated herein by reference in its entirety. Some embodiments ofthe methods and compositions provided herein employ the separate, butparallel workflow concept described in U.S. application Ser. No.17/147,272; for example, in some embodiments, a secreted factor-bindingreagent specific oligonucleotide (e.g., an antibody oligonucleotide) ishybridized to an oligonucleotide barcode and extended to enable aseparate, but parallel workflow for secreted factor quantitation andmRNA quantitation from the same beads.

In some embodiments of the methods and compositions provided herein, theoligonucleotide barcode comprises a cleavage region (comprising, forexample, one or more cleavage sites such as a non-canonical nucleotide(e.g., deoxyuridine) or a restriction enzyme recognition sequence) asdescribed in U.S. application Ser. No. 17/147,283, the content of whichis incorporated herein by reference in its entirety.

FIG. 6 shows a non-limiting exemplary design of a secreted factorbinding reagent specific oligonucleotide (antibody oligonucleotideillustrated here) that is associated with a secreted factor-bindingreagent (antibody illustrated here). The secreted factor-binding reagentspecific oligonucleotide 604 can be associated with secretedfactor-binding reagent 602 through linker 616. The secretedfactor-binding reagent specific oligonucleotide 604 can be detached fromthe secreted factor-binding reagent 602 using chemical, optical or othermeans. The secreted factor-binding reagent specific oligonucleotide 604can be an mRNA mimic. The secreted factor-binding reagent specificoligonucleotide 604 can include a second universal sequence 606 (e.g., aprimer adapter), a second molecular label 608 (e.g., a unique molecularlabel sequence), an antibody barcode 610 (e.g., a unique factoridentifier sequence), an alignment sequence 612, and a poly(A) tail 614.

FIGS. 4A-4C show a schematic illustration of a non-limiting exemplaryworkflow for measurement of the number of copies of one or more secretedfactors secreted by a single cell. The workflow can comprise analysis ofcells 404 (e.g., T cells, B cells, tumor cells, myeloid cells, bloodcells, normal cells, fetal cells, maternal cells, or a mixture thereof)with first plurality of first solid supports 410 (e.g., beads). Theworkflow can comprise contacting cells 404 with first solid supports410. The workflow can comprise partitioning 400 a the cells 404 and thefirst solid supports 410 to a plurality of first partitions 402. A firstpartition 402 (e.g., a well, a droplet) of the plurality of firstpartitions can comprise a single cell 404 and a single first solidsupport 410. A cell 404 can comprise secretory vesicles 406 comprisingunreleased secretory factors 408. A cell 404 can capable of secreting aplurality of secreted factors 408. A first solid support 410 cancomprise capture probes 412 capable of specifically binding to at leastone of the plurality of secreted factors 408 secreted by a single cell404. The workflow can comprise an incubation 400 b comprising secretionof secreted factors and binding thereof to capture probes. The workflowcan comprise pooling 400 c of the single first solid supports 410 fromeach first partition 402 (to generate a second plurality of first solidsupports). The workflow can comprise contacting 400 d the first solidsupport 410 with a plurality of secreted factor-binding reagents 414each capable of specifically binding to a secreted factor bound by acapture probe. Each of the plurality of secreted factor-binding reagentscan comprise a secreted factor-binding reagent specific oligonucleotide416 comprising a unique factor identifier sequence for the secretedfactor-binding reagent. The workflow can comprise an incubation 400 ecomprising binding of the secreted factor-binding reagents 414 tosecreted factor 408 bound by a capture probe 412. The workflow cancomprise one or more washes 400 f comprising removal of secretedfactor-binding reagents 414 that are not bound to secreted factor 408bound by a capture probe 412 (to generate a third plurality of firstsolid supports). The workflow can comprise partitioning 400 g the firstsolid supports 410 to a plurality of second partitions 418. A secondpartition 418 (e.g., a well, a droplet) of the plurality of secondpartitions can comprise a single first solid support 410 and a singlethird solid support 420. Third solid support 420 can comprise aplurality of oligonucleotide barcodes 422. Oligonucleotide barcodes 422can comprise a first molecular label and/or cellular label. The workflowcan comprise contacting oligonucleotide barcodes 422 with the secretedfactor-binding reagent specific oligonucleotides 416 for hybridization.The workflow can comprise barcoding, library preparation, and/orsequencing 400 h as described herein. For example, the workflow cancomprise extending oligonucleotide barcodes 422 hybridized to thesecreted factor-binding reagent specific oligonucleotides 416 togenerate a plurality of barcoded secreted factor-binding reagentspecific oligonucleotides each comprising a sequence complementary to atleast a portion of the unique factor identifier sequence and the firstmolecular label. The method can comprise obtaining sequence informationof barcoded secreted factor-binding reagent specific oligonucleotides,or products thereof, to determine the number of copies of a secretedfactor 408 secreted by each of the one or more single cells 404. Theworkflow can comprise performing the steps with a plurality of cells(e.g., in bulk).

FIGS. 5A-5C show a schematic illustration of a non-limiting exemplaryworkflow for measuring the number of copies of a secreted factorsecreted by a single cell and the number of copies of a nucleic acidtarget in a single cell. The workflow can comprise analysis of cells 504(e.g., T cells, B cells, tumor cells, myeloid cells, blood cells, normalcells, fetal cells, maternal cells, or a mixture thereof) with firstplurality of second solid supports 510 (e.g., beads). The workflow cancomprise contacting cells 504 with second solid supports 510. Theworkflow can comprise partitioning 500 a the cells 504 and the secondsolid supports 510 to a plurality of first partitions 502. A firstpartition 502 (e.g., a well, a droplet) of the plurality of firstpartitions can comprise a single cell 504 and a single second solidsupport 510. A cell 504 can comprise secretory vesicles 506 comprisingunreleased secretory factors 508. A cell 504 can capable of secreting aplurality of secreted factors 508. A second solid support 510 cancomprise capture probes 512 capable of specifically binding to at leastone of the plurality of secreted factors 508 secreted by a single cell504. Cells 504 can comprise a surface cellular target 524 and copies ofa nucleic acid target 526. The second solid support 510 can compriseanchor probes 528 capable of specifically binding to the surfacecellular target 524. The single cell 504 can be capable of becomingassociated with a second solid support 510 via the anchor probe 528binding to the surface cellular target 524. The workflow can comprise anincubation 500 b comprising secretion of secreted factors and bindingthereof to capture probes and the single cell 504 becoming associatedwith a second solid support 510 via the anchor probe 528 binding to thesurface cellular target 524. The workflow can comprise pooling 500 c ofsingle cells associated with a second solid support from each firstpartition 502 (to generate a first plurality of single cells associatedwith a second solid support). The workflow can comprise contacting 500 dthe single cells associated with a second solid support with a pluralityof secreted factor-binding reagents 514 each capable of specificallybinding to a secreted factor bound by a capture probe. Each of theplurality of secreted factor-binding reagents can comprise a secretedfactor-binding reagent specific oligonucleotide 516 comprising a uniquefactor identifier sequence for the secreted factor-binding reagent. Theworkflow can comprise an incubation 500 e comprising binding of thesecreted factor-binding reagents 514 to secreted factor 508 bound by acapture probe 512. The workflow can comprise one or more washes 500 fcomprising removal of secreted factor-binding reagents 514 that are notbound to secreted factor 508 bound by a capture probe 512 (to generate asecond plurality of single cells associated with a second solidsupport). The workflow can comprise partitioning 500 g single cellsassociated with a second solid support to a plurality of secondpartitions 518. A second partition 518 (e.g., a well, a droplet) of theplurality of second partitions can comprise a single cells associatedwith a second solid support and a single third solid support 520. Thirdsolid support 520 can comprise a plurality of oligonucleotide barcodes522. Oligonucleotide barcodes 522 can comprise a first molecular labeland/or cellular label. The workflow can comprise contactingoligonucleotide barcodes 522 with the secreted factor-binding reagentspecific oligonucleotides 516 for hybridization. The workflow cancomprise cell lysis barcoding, library preparation, and/or sequencing500 h as described herein. For example, the workflow can compriseextending oligonucleotide barcodes 522 hybridized to the secretedfactor-binding reagent specific oligonucleotides 516 to generate aplurality of barcoded secreted factor-binding reagent specificoligonucleotides each comprising a sequence complementary to at least aportion of the unique factor identifier sequence and the first molecularlabel. The workflow can comprise lysing the single cell 504 to releasecopies of the nucleic acid target 526 contained therein. The workflowcan comprise contacting oligonucleotide barcodes 522 with the copies ofthe nucleic acid target 526 for hybridization. The workflow can compriseextending the plurality of oligonucleotide barcodes 522 hybridized tothe copies of a nucleic acid target 526 to generate a plurality ofbarcoded nucleic acid molecules each comprising a sequence complementaryto at least a portion of the nucleic acid target and the first molecularlabel. The method can comprise obtaining sequence information ofbarcoded secreted factor-binding reagent specific oligonucleotides, orproducts thereof, to determine the number of copies of a secreted factor508 secreted by each of the one or more single cells 504. The workflowcan comprise obtaining sequence information of the plurality of barcodednucleic acid molecules, or products thereof, to determine the copynumber of the nucleic acid target 526 in each of the one or more singlecells 504. The workflow can comprise performing the steps with aplurality of cells (e.g., in bulk).

Some embodiments disclosed herein provide a plurality of compositionseach comprising a secreted factor binding reagent (such as a proteinbinding reagent). The secreted factor binding reagent can be conjugatedwith an oligonucleotide, wherein the oligonucleotide comprises a uniquefactor identifier for the secreted factor binding reagent that it isconjugated with. The unique factor identifiers can be, for example, anucleotide sequence having any suitable length, for example, from about4 nucleotides to about 200 nucleotides. In some embodiments, the uniquefactor identifier is a nucleotide sequence of 25 nucleotides to about 45nucleotides in length. In some embodiments, the unique factor identifiercan have a length that is, is about, is less than, is greater than, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90,100, 200 nucleotides, or a range that is between any two of the abovevalues.

In some embodiments, the unique factor identifiers are selected from adiverse set of unique factor identifiers. The diverse set of uniquefactor identifiers can comprise, or comprise about, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,5000, or a number or a range between any two of these values, differentunique factor identifiers. The diverse set of unique factor identifierscan comprise at least, or comprise at most, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000,different unique factor identifiers. In some embodiments, the set ofunique factor identifiers is designed to have minimal sequence homologyto the DNA or RNA sequences of the sample to be analyzed. In someembodiments, the sequences of the set of unique factor identifiers aredifferent from each other, or the complement thereof, by, or by about,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or a number or a rangebetween any two of these values. In some embodiments, the sequences ofthe set of unique factor identifiers are different from each other, orthe complement thereof, by at least, or by at most, 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 nucleotides. In some embodiments, the sequences of the setof unique factor identifiers are different from each other, or thecomplement thereof, by at least 3%, at least 5%, at least 8%, at least10%, at least 15%, at least 20%, or more.

Any suitable secreted factor binding reagents, anchor probes, andcapture probes are contemplated in this disclosure, such as proteinbinding reagents, antibodies or fragments thereof, aptamers, smallmolecules, ligands, peptides, oligonucleotides, etc., or any combinationthereof. The secreted factor binding reagents, anchor probes, and/orcapture probes can be polyclonal antibodies, monoclonal antibodies,recombinant antibodies, single chain antibody (sc-Ab), or fragmentsthereof, such as Fab, Fv, etc. In some embodiments, the plurality ofsecreted factor binding reagents can comprise, or comprise about, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 5000, or a number or a range between any two of thesevalues, different secreted factor binding reagents. In some embodiments,the plurality of secreted factor binding reagents can comprise at least,or comprise at most, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, or 5000, different secreted factorbinding reagents. In some embodiments, the plurality of anchor probescan comprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number ora range between any two of these values, different anchor probes. Insome embodiments, the plurality of anchor probes can comprise at least,or comprise at most, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, or 5000, different anchor probes.In some embodiments, the plurality of capture probes can comprise, orcomprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 5000, or a number or a range between anytwo of these values, different capture probes. In some embodiments, theplurality of capture probes can comprise at least, or comprise at most,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, or 5000, different capture probes.

The oligonucleotide can be conjugated with the secreted factor bindingreagent through various mechanisms. In some embodiments, theoligonucleotide can be conjugated with the secreted factor bindingreagent covalently. In some embodiment, the oligonucleotide can beconjugated with the secreted factor binding reagent non-covalently. Insome embodiments, the oligonucleotide is conjugated with the secretedfactor binding reagent through a linker. The linker can be, for example,cleavable or detachable from the secreted factor binding reagent and/orthe oligonucleotide. In some embodiments, the linker can comprise achemical group that reversibly attaches the oligonucleotide to thesecreted factor binding reagents. The chemical group can be conjugatedto the linker, for example, through an amine group. In some embodiments,the linker can comprise a chemical group that forms a stable bond withanother chemical group conjugated to the secreted factor bindingreagent. For example, the chemical group can be a UV photocleavablegroup, a disulfide bond, a streptavidin, a biotin, an amine, etc. Insome embodiments, the chemical group can be conjugated to the secretedfactor binding reagent through a primary amine on an amino acid, such aslysine, or the N-terminus. Commercially available conjugation kits, suchas the Protein-Oligo Conjugation Kit (Solulink, Inc., San Diego,Calif.), the Thunder-Link® oligo conjugation system (Innova Biosciences,Cambridge, United Kingdom), etc., can be used to conjugate theoligonucleotide to the secreted factor binding reagent.

The oligonucleotide can be conjugated to any suitable site of thesecreted factor binding reagent (e.g., a protein binding reagent), aslong as it does not interfere with the specific binding between thesecreted factor binding reagent and its secreted factor. In someembodiments, the secreted factor binding reagent is a protein, such asan antibody. In some embodiments, the secreted factor binding reagent isnot an antibody. In some embodiments, the oligonucleotide can beconjugated to the antibody anywhere other than the antigen-binding site,for example, the Fc region, the C_(H)1 domain, the C_(H)2 domain, theC_(H)3 domain, the C_(L) domain, etc. Methods of conjugatingoligonucleotides to binding reagents (e.g., antibodies) have beenpreviously disclosed, for example, in U.S. Pat. No. 6,531,283, thecontent of which is hereby expressly incorporated by reference in itsentirety. Stoichiometry of oligonucleotide to secreted factor bindingreagent can be varied. To increase the sensitivity of detecting thesecreted factor binding reagent specific oligonucleotide in sequencing,it may be advantageous to increase the ratio of oligonucleotide tosecreted factor binding reagent during conjugation. In some embodiments,each secreted factor binding reagent can be conjugated with a singleoligonucleotide molecule. In some embodiments, each secreted factorbinding reagent can be conjugated with more than one oligonucleotidemolecule, for example, at least, or at most, 2, 3, 4, 5, 10, 20, 30, 40,50, 100, 1000, or a number or a range between any two of these values,oligonucleotide molecules wherein each of the oligonucleotide moleculecomprises the same, or different, unique factor identifiers. In someembodiments, each secreted factor binding reagent can be conjugated withmore than one oligonucleotide molecule, for example, at least, or atmost, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, oligonucleotidemolecules, wherein each of the oligonucleotide molecule comprises thesame, or different, unique factor identifiers.

In some embodiments, the plurality of secreted factor binding reagentsare capable of specifically binding to a plurality of secreted factorsin a sample, such as a single cell, a plurality of cells, a tissuesample, a tumor sample, a blood sample, or the like. In someembodiments, the plurality of secreted factors can comprise, or compriseabout, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000, or a number ora range between any two of these values, different secreted factors. Insome embodiments, the plurality of secreted factors can comprise atleast, or comprise at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000,10000, different secreted factors.

In some embodiments, the secreted factor binding reagent specificoligonucleotide can comprise a nucleotide sequence of, or a nucleotidesequence of about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, or a number or a range between any two of these values,nucleotides in length. In some embodiments, the secreted factor bindingreagent specific oligonucleotide comprises a nucleotide sequence of atleast, or of at most, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, or 1000, nucleotides in length.

Oligonucleotide-Conjugated Antibodies

Some embodiments disclosed herein provide a plurality of compositionseach comprising a cellular component binding reagent (such as a proteinbinding reagent) that is conjugated with an oligonucleotide, wherein theoligonucleotide comprises a unique identifier for the cellular componentbinding reagent that it is conjugated with. Cellular component bindingreagents (such as barcoded antibodies) and their uses (such as sampleindexing of cells) have been described in U.S. Patent ApplicationPublication No. US2018/0088112 and U.S. Patent Application PublicationNo. US2018/0346970; the content of each of these is incorporated hereinby reference in its entirety. The are provided, in some embodimentsprovided herein, secreted factor-binding reagents capable ofspecifically binding to a secreted factor. Secreted factor-bindingreagents can comprise a secreted factor-binding reagent specificoligonucleotide. There are provided, in some embodiments, methods forsimultaneous quantitative analysis of a plurality of cellular componenttargets (e.g., protein targets) and copies of a secreted factor secretedby a single cell. The methods and systems described herein can be usedwith methods and systems using antibodies associated with (e.g.,attached to or conjugated with) oligonucleotides (also referred toherein as AbOs or AbOligos). Embodiments of using AbOs to determineprotein expression profiles in single cells and tracking sample originshave been described in U.S. patent application Ser. No. 15/715,028,published as U.S. Patent Application Publication No. 2018/0088112, andU.S. patent application Ser. No. 15/937,713; the content of each isincorporated by reference herein in its entirety.

Unique Molecular Label Sequence

In some embodiments, the methods and compositions provided hereincomprise an oligonucleotide associated with a cellular component-bindingreagent (e.g., antibody oligonucleotide (“AbOligo” or “AbO”), bindingreagent oligonucleotide, cellular component-binding reagent specificoligonucleotides, sample indexing oligonucleotides) as described in U.S.application Ser. No. 16/747,737, filed on Jan. 21, 2020, the content ofwhich is incorporated herein by reference in its entirety. In someembodiments, the oligonucleotide associated with a cellularcomponent-binding reagent (e.g., antibody oligonucleotide (“AbOligo” or“AbO”), binding reagent oligonucleotide, a secreted factor-bindingreagent specific oligonucleotide, cellular component-binding reagentspecific oligonucleotides, sample indexing oligonucleotides) comprises aunique molecular label sequence (also referred to as a molecular index(MI), “molecular barcode,” or Unique Molecular Identifier (UMI)). Insome embodiments, binding reagent oligonucleotide species comprisingmolecule barcodes as described herein reduce bias by increasingsensitivity, decreasing relative standard error, or increasingsensitivity and/or reducing standard error. The molecule barcode cancomprise a unique sequence, so that when multiple sample nucleic acids(which can be the same and/or different from each other) are associatedone-to-one with molecule barcodes, different sample nucleic acids candifferentiated from each other by the molecule barcodes. As such, evenif a sample comprises two nucleic acids having the same sequence, eachof these two nucleic acids can be labeled with a different moleculebarcode, so that nucleic acids in the population can be quantified, evenafter amplification. The molecule barcode can comprise a nucleic acidsequence of at least 5 nucleotides, for example at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, or 50 nucleotides, including ranges between any two ofthe listed values, for example 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20,5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45,6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9,6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13,7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20,8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30,9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40,10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11nucleotides. In some embodiments, the nucleic acid sequence of themolecule barcode comprises a unique sequence, for example, so that eachunique oligonucleotide species in a composition comprises a differentmolecule barcode. In some embodiments, two or more uniqueoligonucleotide species can comprise the same molecule barcode, butstill differ from each other. For example, if the unique oligonucleotidespecies include sample barcodes, each unique oligonucleotide specieswith a particular sample barcode can comprise a different moleculebarcode. In some embodiments, a composition comprising uniqueoligonucleotide species comprises a molecule barcode diversity of atleast 1000 different molecule barcodes, and thus at least 1000 uniqueoligonucleotide species. In some embodiments, a composition comprisingunique oligonucleotide species comprises a molecule barcode diversity ofat least 6,500 different molecule barcodes, and thus at least 6,500unique oligonucleotide species. In some embodiments, a compositioncomprising unique oligonucleotide species comprises a molecule barcodediversity of at least 65,000 different molecule barcodes, and thus atleast 65,000 unique oligonucleotide species.

In some embodiments, the unique molecular label sequence is positioned5′ of the unique identifier sequence without any intervening sequencesbetween the unique molecular label sequence and the unique identifiersequence. In some embodiments, the unique molecular label sequence ispositioned 5′ of a spacer, which is positioned 5′ of the uniqueidentifier sequence, so that a spacer is between the unique molecularlabel sequence and the unique identifier sequence. In some embodiments,the unique identifier sequence is positioned 5′ of the unique molecularlabel sequence without any intervening sequences between the uniqueidentifier sequence and the unique molecular label sequence. In someembodiments, the unique identifier sequence is positioned 5′ of aspacer, which is positioned 5′ of the unique molecular label sequence,so that a spacer is between the unique identifier sequence and theunique molecular label sequence.

The unique molecular label sequence can comprise a nucleic acid sequenceof at least 3 nucleotides, for example at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 nucleotides, including ranges between any two of thelisted values, for example 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20,3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-50,4-45, 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10,4-9, 4-8, 4-7, 4-6, 4-5, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15,5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45, 6-40,6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8,6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12,7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15,8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25,9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35,10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides.In some embodiments, the unique molecular label sequence is 2-20nucleotides in length.

In some embodiments, the unique molecular label sequence of the bindingreagent oligonucleotide comprises the sequence of at least three repeatsof the doublets “VN” and/or “NV” (in which each “V” is any of A, C, orG, and in which “N” is any of A, G, C, or T), for example at least 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats,including ranges between any two of the listed values. Examples ofmultiple repeats of the doublet “VN” include VN, VNVN, VNVNVN, andVNVNVNVN. It is noted that while the formulas “VN” and “NV” describeconstraints on the base content, not every V or every N has to be thesame or different. For example, if the molecule barcodes of uniqueoligonucleotide species in a composition comprised VNVNVN, one moleculebarcode can comprise the sequence ACGGCA, while another molecule barcodecan comprise the sequence ATACAT, while another molecule barcode couldcomprise the sequence ATACAC. It is noted that any number of repeats ofthe doublet “VN” would have a T content of no more than 50%. In someembodiments, at least 95% of the unique oligonucleotide species of acomposition comprising at least 1000 unique oligonucleotide speciescomprise molecule barcodes comprising at least three repeats of thedoublets “VN” and/or “NV,” for example at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, including rangesbetween any two of the listed values. In some embodiments, at least 99%of the unique oligonucleotide species of a composition comprising atleast 1000 unique oligonucleotide species comprise molecule barcodescomprising at least three repeats of the doublets “VN” and/or “NV,” forexample at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 repeats, including ranges between any two of the listedvalues. In some embodiments, at least 99.9% of the uniqueoligonucleotide species of a composition comprising at least 1000 uniqueoligonucleotide species comprise molecule barcodes comprising at leastthree repeats of the doublets “VN” and/or “NV,” for example at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats,including ranges between any two of the listed values. In someembodiments, at least 95% of the unique oligonucleotide species of acomposition comprising at least 6500 unique oligonucleotide speciescomprise molecule barcodes comprising at least three repeats of thedoublets “VN” and/or “NV,” for example at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, including rangesbetween any two of the listed values. In some embodiments, at least 99%of the unique oligonucleotide species of a composition comprising atleast 6500 unique oligonucleotide species comprise molecule barcodescomprising at least three repeats of the doublets “VN” and/or “NV,” forexample at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 repeats, including ranges between any two of the listedvalues. In some embodiments, at least 99.9% of the uniqueoligonucleotide species of a composition comprising at least 6500 uniqueoligonucleotide species comprise molecule barcodes comprising at leastthree repeats of the doublets “VN” and/or “NV,” for example at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats,including ranges between any two of the listed values. In someembodiments, at least 95% of the unique oligonucleotide species of acomposition comprising at least 65,000 unique oligonucleotide speciescomprise molecule barcodes comprising at least three repeats of thedoublets “VN” and/or “NV,” for example at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, including rangesbetween any two of the listed values. In some embodiments, at least 99%of the unique oligonucleotide species of a of composition comprising atleast 65,000 unique oligonucleotide species comprise molecule barcodescomprising at least three repeats of the doublets “VN” and/or “NV,” forexample at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 repeats, including ranges between any two of the listedvalues. In some embodiments, at least 99.9% of the uniqueoligonucleotide species of a composition comprising at least 65,000unique oligonucleotide species comprise molecule barcodes comprising atleast three repeats of the doublets “VN” and/or “NV,” for example atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20repeats, including ranges between any two of the listed values. In someembodiments, the composition consists of or consists essentially of atleast 1000, 6500, or 65,000 unique oligonucleotide species that eachhave a molecule barcode comprising the sequence VNVNVN. In someembodiments, the composition consists of or consists essentially of atleast 1000, 6500, or 65,000 unique oligonucleotide species that each hasa molecule barcode comprising the sequence VNVNVNVN. In someembodiments, at least 95%, 99%, or 99.9% of the barcode regions of thecomposition as described herein comprise at least three repeats of thedoublets “VN” and/or “NV,” as described herein. In some embodiments,unique molecular label sequences comprising repeated “doublets “VN”and/or “NV” can yield low bias, while providing a compromise betweenreducing bias and maintaining a relatively large quantity of availablenucleotide sequences, so that relatively high diversity can be obtainedin a relatively short sequence, while still minimizing bias. In someembodiments, unique molecular label sequences comprising repeated“doublets “VN” and/or “NV” can reduce bias by increasing sensitivity,decreasing relative standard error, or increasing sensitivity andreducing standard error. In some embodiments, unique molecular labelsequences comprising repeated “doublets “VN” and/or “NV” improveinformatics analysis by serving as a geomarker. In some embodiments, therepeated doublets “VN” and/or “NV” described herein reduce the incidenceof homopolymers within the unique molecular label sequences. In someembodiments, the repeated doublets “VN” and/or “NV” described hereinbreak up homopolymers.

In some embodiments, the sample indexing oligonucleotide comprises afirst molecular label sequence. In some embodiments, the first molecularlabel sequences of at least two sample indexing oligonucleotides aredifferent, and the sample indexing sequences of the at least two sampleindexing oligonucleotides are identical. In some embodiments, the firstmolecular label sequences of at least two sample indexingoligonucleotides are different, and the sample indexing sequences of theat least two sample indexing oligonucleotides are different. In someembodiments, the cellular component-binding reagent specificoligonucleotide comprises a second molecular label sequence. In someembodiments, the second molecular label sequences of at least twocellular component-binding reagent specific oligonucleotides aredifferent, and the unique identifier sequences of the at least twocellular component-binding reagent specific oligonucleotides areidentical. In some embodiments, the second molecular label sequences ofat least two cellular component-binding reagent specificoligonucleotides are different, and the unique identifier sequences ofthe at least two cellular component-binding reagent specificoligonucleotides are different. In some embodiments, the number ofunique second molecular label sequences associated with the uniqueidentifier sequence for the cellular component-binding reagent capableof specifically binding to the at least one cellular component target inthe sequencing data indicates the number of copies of the at least onecellular component target in the one or more of the plurality of cells.In some embodiment, a combination (e.g., minimum, average, and maximum)of (1) the number of unique first molecular label sequences associatedwith the unique identifier sequence for the cellular component-bindingreagent capable of specifically binding to the at least one cellularcomponent target in the sequencing data and (2) the number of uniquesecond molecular label sequences associated with the unique identifiersequence for the cellular component-binding reagent capable ofspecifically binding to the at least one cellular component target inthe sequencing data indicates the number of copies of the at least onecellular component target in the one or more of the plurality of cells.

Alignment Sequence

In some embodiments, the binding reagent oligonucleotide comprises analignment sequence (e.g., the alignment sequence 825bb) adjacent to thepoly(dA) region. The alignment sequence can be 1 or more nucleotides inlength. The alignment sequence can be 2 nucleotides in length. Thealignment sequence can comprise a guanine, a cytosine, a thymine, auracil, or a combination thereof. The alignment sequence can comprise apoly(dT) region, a poly(dG) region, a poly(dC) region, a poly(dU)region, or a combination thereof. In some embodiments, the alignmentsequence is 5′ to the poly(dA) region. Advantageously, in someembodiments, the presence of the alignment sequence enables the poly(A)tail of each of the binding reagent oligonucleotides to have the samelength, leading to greater uniformity of performance. In someembodiments, the percentage of binding reagent oligonucleotides with anidentical poly(dA) region length within a plurality of binding reagentoligonucleotides, each of which comprise an alignment sequence, can be,or be about, 80%, 90%, 91%, 93%, 95%, 97%, 99.9%, 99.9%, 99.99%, or100%, or a number or a range between any two of these values. In someembodiments, the percentage of binding reagent oligonucleotides with anidentical poly(dA) region length within the plurality of binding reagentoligonucleotides, each of which comprise an alignment sequence, can beat least, or be at most, 80%, 90%, 91%, 93%, 95%, 97%, 99.9%, 99.9%,99.99%, or 100%.

The length of the alignment sequence can be different in differentimplementations. In some embodiments, the length of the alignmentsequence can be, or can be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or anumber or a range between any two of these values. In some embodiments,the length of the alignment sequence can be at least, or can be at most,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100. The number of guanine(s),cytosine(s), thymine(s), or uracil(s) in the alignment sequence can bedifferent in different implementations. The number of guanine(s),cytosine(s), thymine(s), or uracil(s) can be, or can be about, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, or a number or a range between any two of thesevalues. The number of guanine(s), cytosine(s), thymine(s), or uracil(s)can be at least, or can be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.In some embodiments, the sample indexing oligonucleotide comprises analignment sequence. In some embodiments, the cellular component-bindingreagent specific oligonucleotide and/or secreted factor-binding reagentspecific oligonucleotide comprises an alignment sequence.

Linker

The binding reagent oligonucleotide (e.g., secreted factor-bindingreagent specific oligonucleotide) can be conjugated with the cellularcomponent binding reagent through various mechanisms. In someembodiments, the binding reagent oligonucleotide can be conjugated withthe cellular component binding reagent covalently. In some embodiments,the binding reagent oligonucleotide can be conjugated with the cellularcomponent binding reagent non-covalently. In some embodiments, thebinding reagent oligonucleotide is conjugated with the cellularcomponent binding reagent through a linker. In some embodiments, thebinding reagent oligonucleotide can comprise the linker. The linker cancomprise a chemical group. The chemical group can be reversibly, orirreversibly, attached to the molecule of the cellular component bindingreagent. The chemical group can be selected from the group consisting ofa UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, and any combination thereof. The linker can comprise a carbonchain. The carbon chain can comprise, for example, 5-50 carbon atoms.The carbon chain can have different numbers of carbon atoms in differentembodiments. In some embodiments, the number of carbon atoms in thecarbon chain can be, or can be about, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, or a number or a range between any two of these values. In someembodiments, the number of carbon atoms in the carbon chain can be atleast, or can be at most, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.In some embodiments, the carbon chain comprises 2-30 carbons. In someembodiments, the carbon chain comprises 12 carbons. In some embodiments,amino modifiers employed for binding reagent oligonucleotide can beconjugated to the cellular component binding reagent. In someembodiments, the linker comprises 5′ amino modifier C6 (5AmMC6). In someembodiments, the linker comprises 5′ amino modifier C12 (5AmMC12). Insome embodiments, the linker comprises a derivative of 5AmMC12. In someembodiments, a longer linker achieves a higher efficiency ofconjugation. In some embodiments, a longer linker achieves a higherefficiency of modification prior to conjugation. In some embodiments,increasing the distance between the functional amine and the DNAsequence yields a higher efficiency of conjugation. In some embodiments,increasing the distance between the functional amine and the DNAsequence yields a higher efficiency of modification prior toconjugation. In some embodiments, the use of 5AmMC12 as a linker yieldsa higher efficiency of modification (prior to conjugation) than the useof 5AmMC6 as a linker. In some embodiments the use of 5AmMC12 as alinker yields a higher efficiency of conjugation than the use of 5AmMC6as a linker. In some embodiments, the sample indexing oligonucleotide isassociated with the cellular component-binding reagent through a linker.In some embodiments, the cellular component-binding reagent specificoligonucleotide and/or secreted factor-binding reagent specificoligonucleotide is associated with the cellular component-bindingreagent through a linker.

Antibody-Specific Barcode Sequence

Disclosed herein, in several embodiments, are improvements to the designof the unique identifier sequence (e.g., antibody-specific barcodesequence) of a binding reagent oligonucleotide (e.g., secretedfactor-binding reagent specific oligonucleotide). In some embodimentsthe unique identifier sequence (e.g, sample indexing sequence, uniquefactor identifier sequence, a unique identifier sequence of a cellularcomponent-binding reagent specific oligonucleotide) is designed to havea Hamming distance greater than 3. In some embodiments, the Hammingdistance of the unique identifier sequence can be, or be about, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or a number or a range between any two of thesevalues. In some embodiments, the unique identifier sequences has a GCcontent in the range of 40% to 60% and does not have a predictedsecondary structure (e.g., hairpin). In some embodiments, the uniqueidentifier sequence does not comprise any sequences predicted in silicoto bind to the mouse and/or human transcripts. In some embodiments, theunique identifier sequence does not comprise any sequences predicted insilico to bind to Rhapsody™ and/or SCMK system primers. In someembodiments, the unique identifier sequence does not comprisehomopolymers.

Primer Adapter

In some embodiments, the binding reagent oligonucleotide (e.g., secretedfactor-binding reagent specific oligonucleotide) comprises a primeradapter. In some embodiments, the primer adapter comprises the sequenceof a first universal primer, a complimentary sequence thereof, a partialsequence thereof, or a combination thereof. In some embodiments, thefirst universal primer comprises an amplification primer, acomplimentary sequence thereof, a partial sequence thereof, or acombination thereof. In some embodiments, the first universal primercomprises a sequencing primer, a complimentary sequence thereof, apartial sequence thereof, or a combination thereof. In some embodiments,the sequencing primer comprises an Illumina sequencing primer. In someembodiments, the sequencing primer comprises a portion of an Illuminasequencing primer. In some embodiments, the sequencing primer comprisesa P7 sequencing primer or a portion of P7 sequencing primer. In someembodiments, the primer adapter comprises an adapter for Illumina P7 ora partial adapter for Illumina P7. In some embodiments, theamplification primer is an Illumina P7 sequence or a subsequencethereof. In some embodiments, the sequencing primer is an Illumina R2sequence or a subsequence thereof. In some embodiments, the firstuniversal primer is 5-50 nucleotides in length. In some embodiments, Theprimer adapter can comprise a nucleic acid sequence of at least 5nucleotides, for example at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides, including ranges between any two of the listed values, forexample 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13,5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45, 6-40, 6-35, 6-30,6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50,7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10,7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14, 8-13,8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15,9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25,10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides. The primeradapter can comprise a nucleic acid sequence of at least 5 nucleotidesof the sequence of a first universal primer, an amplification primer, asequencing primer, a complimentary sequence thereof, a partial sequencethereof, or a combination thereof, for example at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, or 50 nucleotides, including ranges between any two ofthe listed values, for example 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20,5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45,6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9,6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13,7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20,8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30,9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40,10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11nucleotides of the sequence of a first universal primer, anamplification primer, a sequencing primer, a complimentary sequencethereof, a partial sequence thereof, or a combination thereof

A conventional amplification workflow for sequencing library preparationcan employ three rounds of PCR, such as, for example: a first round(“PCR 1”) employing a target-specific primer and a primer against theuniversal Illumina sequencing primer 1 sequence; a second round (“PCR2”) using a nested target-specific primer flanked by Illumina sequencingprimer 2 sequence, and a primer against the universal Illuminasequencing primer 1 sequence; and a third round (“PCR 3”) addingIllumina P5 and P7 and sample index. Advantageously, in someembodiments, the primer adapter disclosed herein enables a shorter andsimpler workflow in library preparation as compared to if the startingtemplate (e.g., a sample indexing oligonucleotide attached to a bead)does not have a primer adapter. In some embodiments, the primer adapterreduces pre-sequencing PCR amplification of a template by one round (ascompared to if the template does not comprise a primer adapter). In someembodiments, the primer adapter reduces pre-sequencing PCR amplificationof the template to one round (as compared to if the template does notcomprise a primer adapter). In some embodiments, a template comprisingthe primer adapter does not require a PCR amplification step forattachment of Illumina sequencing adapters that would requirepre-sequencing if the template did not comprise a primer adapter. Insome embodiments, the primer adapter sequence (or a subsequence thereof)is not part of the sequencing readout of a sequencing templatecomprising a primer adapter sequence and therefore does not affect readquality of a template comprising a primer adapter. In some embodiments,a template comprising the primer adapter has decreased sequencingdiversity as compared to if the template does not comprise a primeradapter.

In some embodiments, the sample indexing oligonucleotide comprises aprimer adapter. In some embodiments, replicating a sample indexingoligonucleotide, a barcoded sample indexing oligonucleotide, or aproduct thereof, comprises using a first universal primer, a firstprimer comprising the sequence of the first universal primer, or acombination thereof, to generate a plurality of replicated sampleindexing oligonucleotides. In some embodiments, replicating a sampleindexing oligonucleotide, a barcoded sample indexing oligonucleotide, ora product thereof, comprises using a first universal primer, a firstprimer comprising the sequence of the first universal primer, a seconduniversal primer, a second primer comprising the sequence of the seconduniversal primer, or a combination thereof, to generate the plurality ofreplicated sample indexing oligonucleotides. In some embodiments, thecellular component-binding reagent specific oligonucleotide and/orsecreted factor-binding reagent specific oligonucleotide comprises aprimer adapter, the sequence of a first universal primer, acomplementary sequence thereof, a partial sequence thereof, or acombination thereof.

Binding Reagent Oligonucleotide Barcoding

FIG. 8 shows a schematic illustration of a non-limiting exemplaryworkflow of barcoding of a binding reagent oligonucleotide 825 (antibodyoligonucleotide illustrated here, e.g., secreted factor-binding reagentspecific oligonucleotide) that is associated with a binding reagent 805(antibody illustrated here, such as, for example, a secretedfactor-binding reagent). The binding reagent oligonucleotide 825 can beassociated with binding reagent 805 through linker 8251. The bindingreagent oligonucleotide 825 can be detached from the binding reagentusing chemical, optical or other means. The binding reagentoligonucleotide 825 can be an mRNA mimic. The binding reagentoligonucleotide 825 can include a primer adapter 825 pa, an antibodymolecular label 825 am (e.g., a unique molecular label sequence), anantibody barcode 825 ab (e.g., a unique identifier sequence), analignment sequence 825 bb, and a poly(A) tail 825 a. In someembodiments, the primer adapter 825 pa comprises the sequence of a firstuniversal primer, a complimentary sequence thereof, a partial sequencethereof, or a combination thereof. In some embodiments, the primeradapter 825 pa can be the same for all or some of binding reagentoligonucleotides 825. In some embodiments, the antibody barcode 825 abcan be the same for all or some of binding reagent oligonucleotides 825.In some embodiments, the antibody barcode 825 ab of different bindingreagent oligonucleotides 825 are different. In some embodiments, theantibody molecular label 825 am of different binding reagentoligonucleotides 825 are different.

The binding reagent oligonucleotides 825 can be barcoded using aplurality of barcodes 815 (e.g., barcodes 815 associated with aparticle, such as a bead 810) to create a plurality of barcoded bindingreagent oligonucleotides 840. In some embodiments, a barcode 815 caninclude a poly(dT) region 815 t for binding to a binding reagentoligonucleotide 825, optionally a molecular label 815 m (e.g., fordetermining the number of occurrences of the binding reagentoligonucleotides), a cell label 815 c, and a universal label 815 u. Insome embodiments the barcode 815 is hybridized to the poly(dT) region815 t of binding reagent oligonucleotides 825. In some embodimentsbarcoded binding reagent oligonucleotides 840 are generated by extending(e.g., by reverse transcription) the barcode 815 hybridized to thebinding reagent oligonucleotide 825. In some embodiments, barcodedbinding reagent oligonucleotides 840 comprise primer adapter 825 pa, anantibody molecular label 825 am (e.g., a unique molecular labelsequence), an antibody barcode 825 ab (e.g., a unique identifiersequence), an alignment sequence 825 bb, poly(dT) region 815 t,molecular label 815 m, cell label 815 c, and universal label 815 u.

In some embodiments, the barcoded binding reagent oligonucleotidesdisclosed herein comprises two unique molecular label sequences: amolecular label sequence derived from the barcode (e.g., molecular label815 m) and a molecular label sequence derived from a binding reagentoligonucleotide (e.g., antibody molecular label 825 am, the firstmolecular label sequence of a sample indexing oligonucleotide, thesecond molecular label sequence of a cellular component-binding reagentspecific oligonucleotide, the molecular label of a secretedfactor-binding reagent specific oligonucleotide). As used herein, “dualmolecular indexing” refers to methods and compositions disclosed hereinemploying barcoded binding reagent oligonucleotides (or productsthereof) that comprise a first unique molecular label sequence andsecond unique molecular label sequence (or complementary sequencesthereof). In some embodiments, the methods of sample identification andof quantitative analysis of cellular component targets disclosed hereincan comprise obtaining the sequence of information of the barcodemolecular label sequence and/or the binding reagent oligonucleotidemolecular label sequence. In some embodiments, the number of barcodemolecular label sequences associated with the unique identifier sequencefor the cellular component-binding reagent capable of specificallybinding to the at least one cellular component target in the sequencingdata indicates the number of copies of the at least one cellularcomponent target in the one or more of the plurality of cells. In someembodiments, the number of binding reagent oligonucleotide molecularlabel sequences associated with the unique identifier sequence for thecellular component-binding reagent capable of specifically binding tothe at least one cellular component target in the sequencing dataindicates the number of copies of the at least one cellular componenttarget in the one or more of the plurality of cells. In someembodiments, the number of both the binding reagent oligonucleotidemolecular label sequences and barcode molecular label sequencesassociated with the unique identifier sequence for the cellularcomponent-binding reagent capable of specifically binding to the atleast one cellular component target in the sequencing data indicates thenumber of copies of the at least one cellular component target in theone or more of the plurality of cells.

The use of PCR to amplify the amount of material before starting thesequencing protocol adds the potential for artifacts, such asartifactual recombination during amplification occurs when prematuretermination products prime a subsequent round of synthesis). In someembodiments, the methods of dual molecular indexing provided hereinallow the identification of PCR chimeras given sufficient sequencingdepth. Additionally, in some embodiments, the addition of the uniquemolecular label sequence to the binding reagent oligonucleotideincreases stochastic labelling complexity. Thus, in some embodiments,the presence of the unique molecular label sequence in the bindingreagent oligonucleotide can overcome UMI diversity limitations. In someembodiments the methods of dual molecular indexing provided hereindecrease the number of cellular component targets flagged as “saturated”during post-sequencing molecular coverage calculations by at least about2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%,250%, 500%, 1000%, or higher and overlapping ranges therein) compared toif the methods and compositions are not used.

Probes, Binding Reagents, and Solid Supports

The first solid support and/or the second solid support can be sized andshaped to approximate a cell. In some embodiments, the first solidsupport and/or the second solid support has the dimensions of a cell(e.g., a mammalian cell, a yeast cell, an insect cell, a plant cell, abacterial cell, or any combination thereof). The first solid support,second solid support, and/or third solid support can comprise asynthetic particle or a planar surface. At least one of the plurality ofoligonucleotide barcodes can be immobilized or partially immobilized onthe synthetic particle. At least one of the plurality of oligonucleotidebarcodes can be enclosed or partially enclosed in the syntheticparticle. The synthetic particle can be disruptable. The syntheticparticle can comprise a bead. The bead can comprise: a sepharose bead, astreptavidin bead, an agarose bead, a magnetic bead, a conjugated bead,a protein A conjugated bead, a protein G conjugated bead, a protein A/Gconjugated bead, a protein L conjugated bead, an oligo(dT) conjugatedbead, a silica bead, a silica-like bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof; a materialselected from the group consisting of polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, sepharose, cellulose, nylon, silicone, and anycombination thereof; or a disruptable hydrogel particle.

In some embodiments, each of the plurality of oligonucleotide barcodescomprises a linker functional group, the synthetic particle comprises asolid support functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

In some embodiments, each of the plurality of anchor probes comprises alinker functional group, the synthetic particle comprises a solidsupport functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

In some embodiments, each of the plurality of capture probes comprises alinker functional group, the synthetic particle comprises a solidsupport functional group, and the support functional group and thelinker functional group are associated with each other. The linkerfunctional group and the support functional group can be individuallyselected from the group consisting of C6, biotin, streptavidin, primaryamine(s), aldehyde(s), ketone(s), and any combination thereof.

The secreted factor-binding reagent and the capture probe can be capableof binding to distinct epitopes of the same secreted factor. In someembodiments, one or more of the secreted factor-binding reagents, thecapture probe, and the anchor probe comprise an antibody or fragmentthereof. The antibody or fragment thereof can comprise a monoclonalantibody. The antibody or fragment thereof can comprise a Fab, a Fab′, aF(ab′)₂, a Fv, a scFv, a dsFv, a diabody, a triabody, a tetrabody, amultispecific antibody formed from antibody fragments, a single-domainantibody (sdAb), a single chain comprising complementary scFvs (tandemscFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linkedFv, a dual variable domain immunoglobulin (DVD-Ig) binding protein or ananobody, an aptamer, an affibody, an affilin, an affitin, an affimer,an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitzdomain peptide, a monobody, or any combination thereof. The captureprobe and/or the anchor probe can be conjugated to the first solidsupport and/or the second solid support by a 1,3-dipolar cycloadditionreaction, a hetero-Diels-Alder reaction, a nucleophilic substitutionreaction, a non-aldol type carbonyl reaction, an addition tocarbon-carbon multiple bond, an oxidation reaction, a click reaction, orany combination thereof.

The at least one secreted factor can comprise a lymphokine, aninterleukin, a chemokine, or any combination thereof. The at least onesecreted factor can comprise a cytokine, a hormone, a molecular toxin,or any combination thereof. The at least one secreted factor cancomprise a nerve growth factor, a hepatic growth factor, a fibroblastgrowth factor, a vascular endothelial growth factor, a platelet-derivedgrowth factor, a transforming growth factor, an osteoinductive factor,an interferon, a colony stimulating factor, or any combination thereof.The at least one secreted factor can comprise angiogenin,angiopoietin-1, angiopoietin-2, bNGF, cathepsin S, Galectin-7, GCP-2,G-CSF, GM-CSF, PAI-1, PDGF-AA, PDGF-BB, PDGF-AB, PlGF, PlGF-2, SDF-1,Tie2, VEGF-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2, VEGF-R3, 6Ckine,angiopoietin-1, angiopoietin-2, BLC, BRAK, CD186, ENA-78, Eotaxin-1,Eotaxin-2, Eotaxin-3, EpCAM, GDF-15, GM-CSF, GRO, HCC-4, I-309, IFN-γ,IL-1α, IL-1β, IL-1R4 (ST2), IL-2, IL-2R, IL-3, IL-3Rα, IL-5, IL-6,IL-6R, IL-7, IL-8, IL-8 RB, IL-11, IL-12, IL-12p40, IL-12p70, IL-13,IL-13 R1, IL-13R2, IL-15, IL-15Rα, IL-16, IL-17, IL-17C, IL-17E, IL-17F,IL-17R, IL-18, IL-18BPa, IL-18 Rα, IL-20, IL-23, IL-27, IL-28, IL-31,IL-33, IP-10, I-TAC, LIF, LIX, LRP6, MadCAM-1, MCP-1, MCP-2, MCP-3,MCP-4, M-CSF, MIF, MIG, MIP-1 gamma, MIP-1α, MIP-1β, MIP-1δ, MIP-3α,MIP-3β, MPIF-1, PARC, PF4, RANTES, Resistin, SCF, SCYB16, TACI, TARC,TSLP, TNF-α, TNF-R1, TRAIL-R4, TREM-1, Activin A, Amphiregulin, Axl,BDNF, BMP4, cathepsin S, EGF, FGF-1, FGF-2, FGF-7, FGF-21, Follistatin,Galectin-7, Gash, GDF-15, HB-EGF, HGF, IGFBP-1, IGFBP-3, LAP, NGF R,NrCAM, NT-3, NT-4, PAI-1, TGF-α, TGF-β, TGF-β3, TRAIL-R4, ADAMTS1,cathepsin S, FGF-2, Follistatin, Galectin-7, GCP-2, GDF-15, IGFBP-6,LIF, MMP-9, pro-MMP9, RANK, RANKL, RANTES, SDF-1, CXCR4, or anycombination thereof.

The surface cellular target can comprise a carbohydrate, a lipid, aprotein, an extracellular protein, a cell-surface protein, a cellmarker, a B-cell receptor, a T-cell receptor, a major histocompatibilitycomplex, a tumor antigen, a receptor, an intracellular protein, or anycombination thereof. The surface cellular target can comprise acarbohydrate, a lipid, a protein, or any combination thereof. Thesurface cellular target can comprise CD1a, CD1b, CD1c, CD1d, CD1e, CD2,CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a,CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15u, CD15s, CD15su,CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26,CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38,CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA,CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d,CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58,CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65,CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71,CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82,CD83, CD84, CD85a, CD85d, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90,CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100,CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109,CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119,CD120a, CD120b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126,CD127, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137,CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146,CD147, CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a,CD156b, CD156c, CD157, CD158e, CD158i, CD158k, CD159a, CD159c, CD160,CD161, CD162, CD163, CD164, CD165, CD166, CD167a, CD167b, CD168, CD169,CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s,CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184,CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDw198,CD199, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208,CD209, CD210, CDw210b, CD212, CD213a1, CD213a2, CD215, CD217a, CD218a,CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228,CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R,CD238, CD239, CD240CE, CD240DCE, CD240D, CD241, CD242, CD243, CD244,CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD266,CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276,CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD289,CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a,CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD307a,CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315,CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326,CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337,CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354,CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366,CD367, CD368, CD369, CD370, CD371, BCMA, a HLA protein,β2-microglobulin, or any combination thereof.

Methods for Simultaneous Single Cell Secretome and TranscriptomeAnalysis

Disclosed herein include methods of measuring the number of copies of asecreted factor secreted by a single cell. The method can comprise:contacting one or more single cells with a first plurality of firstsolid supports, the one or more single cells are capable of secreting aplurality of secreted factors, each first solid support comprises aplurality of capture probes capable of specifically binding to at leastone of the plurality of secreted factors secreted by a single cell. Themethod can comprise: contacting the first solid support with a pluralityof secreted factor-binding reagents each capable of specifically bindingto a secreted factor bound by a capture probe, each of the plurality ofsecreted factor-binding reagents comprises a secreted factor-bindingreagent specific oligonucleotide comprising a unique factor identifiersequence for the secreted factor-binding reagent. The method cancomprise: contacting a plurality of oligonucleotide barcodes with thesecreted factor-binding reagent specific oligonucleotides forhybridization, the oligonucleotide barcodes each comprise a firstmolecular label. The method can comprise: extending the plurality ofoligonucleotide barcodes hybridized to the secreted factor-bindingreagent specific oligonucleotides to generate a plurality of barcodedsecreted factor-binding reagent specific oligonucleotides eachcomprising a sequence complementary to at least a portion of the uniquefactor identifier sequence and the first molecular label. The method cancomprise: obtaining sequence information of the plurality of barcodedsecreted factor-binding reagent specific oligonucleotides, or productsthereof, to determine the number of copies of the at least one secretedfactor secreted by each of the one or more single cells. In someembodiments, the one or more single cells comprises T cells, B cells,tumor cells, myeloid cells, blood cells, normal cells, fetal cells,maternal cells, or a mixture thereof.

In some embodiments, contacting one or more single cells with a firstplurality of first solid supports comprises: partitioning the one ormore single cells and the first plurality of first solid supports to aplurality of first partitions, a first partition of the plurality offirst partitions comprises a single cell of the one or more single cellsand a single first solid support of the first plurality of first solidsupports.

In some embodiments, the method comprises, prior to contacting the firstsolid support with a plurality of secreted factor-binding reagents:pooling the single first solid supports from each first partition of theplurality of first partitions to generate a second plurality of firstsolid supports. In some embodiments, contacting the first solid supportwith a plurality of secreted factor-binding reagents comprisescontacting the second plurality of first solid supports with theplurality of secreted factor-binding reagents.

In some embodiments, the method comprises, after contacting the secondplurality of first solid supports with the plurality of secretedfactor-binding reagents, removing one or more secreted factor-bindingreagents of the plurality of secreted factor-binding reagents that arenot contacted with the second plurality of first solid supports togenerate a third plurality of first solid supports. In some embodiments,removing the one or more secreted factor-binding reagents not contactedwith the second plurality of first solid supports comprises: removingthe one or more secreted factor-binding reagents not contacted with therespective at least one of the secreted factor bound by a capture probe.

In some embodiments, contacting a plurality of oligonucleotide barcodeswith the secreted factor-binding reagent specific oligonucleotides forhybridization comprises: partitioning the third plurality of first solidsupports to a plurality of second partitions, a second partition of theplurality of second partitions comprises a single first solid supportfrom the third plurality of first solid supports; and in the secondpartition comprising the single first solid support, contacting aplurality of oligonucleotide barcodes with the secreted factor-bindingreagent specific oligonucleotides for hybridization.

Disclosed herein include methods of measuring the number of copies of asecreted factor secreted by a single cell and the number of copies of anucleic acid target in a single cell. The method can comprise:contacting one or more single cells with a first plurality of secondsolid supports to form one or more single cells associated with a secondsolid support, the one or more single cells comprise a surface cellulartarget and copies of a nucleic acid target, the one or more single cellsare capable of secreting a plurality of secreted factors, each secondsolid support comprises a plurality of capture probes and a plurality ofanchor probes, each of the plurality of anchor probes is capable ofspecifically binding to the surface cellular target, and the captureprobe is capable of specifically binding to at least one of theplurality of secreted factors secreted by a single cell. The method cancomprise: contacting the one or more single cells associated with asecond solid support with a plurality of secreted factor-bindingreagents capable of specifically binding to a secreted factor bound by acapture probe, each of the plurality of secreted factor-binding reagentscomprises a secreted factor-binding reagent specific oligonucleotidecomprising a unique factor identifier sequence for the secretedfactor-binding reagent. The method can comprise: contacting a pluralityof oligonucleotide barcodes with the secreted factor-binding reagentspecific oligonucleotides and the copies of the nucleic acid target forhybridization, the oligonucleotide barcodes each comprise a firstmolecular label. The method can comprise: extending the plurality ofoligonucleotide barcodes hybridized to the copies of a nucleic acidtarget to generate a plurality of barcoded nucleic acid molecules eachcomprising a sequence complementary to at least a portion of the nucleicacid target and the first molecular label. The method can comprise:extending the plurality of oligonucleotide barcodes hybridized to thesecreted factor-binding reagent specific oligonucleotides to generate aplurality of barcoded secreted factor-binding reagent specificoligonucleotides each comprising a sequence complementary to at least aportion of the unique factor identifier sequence and the first molecularlabel. The method can comprise: obtaining sequence information of theplurality of barcoded nucleic acid molecules, or products thereof, todetermine the copy number of the nucleic acid target in each of the oneor more single cells. The method can comprise: obtaining sequenceinformation of the plurality of barcoded secreted factor-binding reagentspecific oligonucleotides, or products thereof, to determine the numberof copies of the at least one secreted factor secreted by each of theone or more single cells. In some embodiments, the one or more singlecells comprises T cells, B cells, tumor cells, myeloid cells, bloodcells, normal cells, fetal cells, maternal cells, or a mixture thereof.

In some embodiments, contacting one or more single cells with a firstplurality of second solid supports to form one or more single cellsassociated with a second solid support comprises: partitioning the oneor more single cells and the plurality of second solid supports to aplurality of first partitions, a first partition of the plurality offirst partitions comprises a single cell of the one or more single cellsand a single second solid support of the plurality of second solidsupports, the single cell is capable of becoming associated with asecond solid support via the anchor probe binding to the surfacecellular target.

In some embodiments, the method comprises, prior to contacting the oneor more single cells associated with a second solid support with aplurality of secreted factor-binding reagents: pooling the single cellsassociated with a second solid support from each first partition of theplurality of first partitions to generate a first plurality of singlecells associated with a second solid support. In some embodiments,contacting the one or more single cells associated with a second solidsupport with a plurality of secreted factor-binding reagents comprisescontacting the first plurality of single cells associated with a secondsolid support with the plurality of secreted factor-binding reagents.

In some embodiments, the method comprises, after contacting the firstplurality of single cells associated with a second solid support withthe plurality of secreted factor-binding reagents, removing one or moresecreted factor-binding reagents of the plurality of secretedfactor-binding reagents that are not contacted with the first pluralityof single cells associated with a second solid support to generate asecond plurality of single cells associated with a second solid support.In some embodiments, removing the one or more secreted factor-bindingreagents not contacted with the first plurality of single cellsassociated with a second solid support comprises: removing the one ormore secreted factor-binding reagents not contacted with the respectiveat least one of the secreted factor bound by a capture probe.

The method can comprise: prior to contacting a plurality ofoligonucleotide barcodes with the secreted factor-binding reagentspecific oligonucleotides and the copies of the nucleic acid target forhybridization: partitioning the second plurality of single cellsassociated with a second solid support to a plurality of secondpartitions, a second partition of the plurality of second partitionscomprises a single cell and a single second solid support from thesecond plurality of single cells associated with a second solid support;in the second partition comprising the single cell and the single secondsolid support, contacting a plurality of oligonucleotide barcodes withthe secreted factor-binding reagent specific oligonucleotides and thecopies of the nucleic acid target for hybridization. The method cancomprise lysing the single cell in the second partition. Lysing thesingle cell can comprise heating the sample, contacting the sample witha detergent, changing the pH of the sample, or any combination thereof.

FIGS. 7A-7D show a schematic illustration of a non-limiting exemplaryworkflow for simultaneous measurement of the number of copies of asecreted factor and a nucleic acid target. A barcode (e.g., a stochasticbarcode, an oligonucleotide barcode 702) can comprise a target bindingregion (e.g., a poly(dT) 710) that can bind to nucleic acid targets(e.g., poly-adenylated RNA transcripts 714 or other nucleic acidtargets, such as for example, secreted factor-binding reagent specificoligonucleotide 720, whether associated with antibodies or havedissociated from antibodies) via a poly(dA) tail 718, or other nucleicacid targets, for labeling or barcoding (e.g., unique labeling). Thetarget-binding region can comprise a gene-specific sequence, anoligo(dT) sequence, a random multimer, or any combination thereof. Theoligonucleotide barcode 702 can also comprise a number of labels. Theoligonucleotide barcode 702 can include first molecular label (ML) 708and a sample label (e.g, partition label, cell label (CL) 706) forlabeling the transcripts and/or tracking sample origins of the RNAtranscripts (or nucleic acid targets, such as for example, antibodyoligonucleotides, whether associated with antibodies or have dissociatedfrom antibodies), respectively, along with one or more additionalsequences flanking the first molecular label 708/cell label 706 regionof each barcode 702 for subsequent reactions, such as, for example, afirst universal sequence 704 (e.g., Read 1 sequence). The repertoire ofsequences of the molecular labels in the oligonucleotide barcodes persample can be sufficiently large for stochastic labeling of RNAtranscripts. The sample label can be, for example, a partition label,and/or a cell label. In some embodiments the barcode is associated witha solid support (e.g., a particle 712). A plurality of barcodes 702 canbe associated with particle 712. In some embodiments, the particle is abead. The bead can be a polymeric bead, for example a deformable bead ora gel bead, functionalized with barcodes or stochastic barcodes (such asgel beads from 10X Genomics (San Francisco, Calif.)). In someimplementation, a gel bead can comprise a polymer-based gels. Gel beadscan be generated, for example, by encapsulating one or more polymericprecursors into droplets. Upon exposure of the polymeric precursors toan accelerator (e.g., tetramethylethylenediamine (TEMED)), a gel beadmay be generated. Poly-adenylated RNA transcripts 714 can comprise RNAsequence 716 r and poly(dA) tail 718. Secreted factor-binding reagentspecific oligonucleotide 720 can comprise a second universal sequence722, a molecular label (e.g., a second molecular label 724) a uniquefactor identifier sequence 726, a sequence complementary to the targetbinding region (e.g., a poly(A) tail 728), or complements thereof. Insome embodiments secreted factor binding reagent specificoligonucleotide 720 is associated with a secreted factor-binding reagent(e.g., antibody 730).

The workflow can comprise hybridization 700 a of the secreted factorbinding reagent specific oligonucleotide 720 and oligonucleotide barcode702. The workflow can comprise hybridization 700 a of thepoly-adenylated RNA transcript 714 and oligonucleotide barcode 702. Theworkflow can comprise extending 700 b the oligonucleotide barcode 702hybridized to the secreted factor binding reagent specificoligonucleotide 720 to generate a barcoded secreted factor bindingreagent specific oligonucleotide 734 comprising a complement of theunique factor identifier sequence 726 rc, a complement of the secondmolecular label 724 rc, and a complement of the second universalsequence 722 rc. In some embodiments, the extension reaction 700 b cancomprise extending the oligonucleotide barcode 702 hybridized to thepoly-adenylated RNA transcript 714 to generate a barcoded nucleic acidmolecule 736 comprising cDNA 1416 c (the reverse complementary sequenceof RNA sequence 716 r). The workflow can comprise denaturation 700 c(e.g., with use of heating and/or chemicals). The workflow can comprisedownstream 700 d primer extension, amplification and/or sequencing ofbarcoded secreted factor binding reagent specific oligonucleotides asdescribed herein. The workflow can comprise downstream 700 e primerextension, amplification and/or sequencing of barcoded cDNAs asdescribed herein.

Barcoded secreted factor binding reagent specific oligonucleotide 734can serve as a template for one or more extension reactions (e.g.,random priming and extension) and/or amplification reactions (e.g.,PCR). For example, barcoded secreted factor binding reagent specificoligonucleotide 734 can undergo a first round of amplification (“PCR1”)700 f employing amplification primers 738 and 740 that can anneal tofirst universal sequence and second universal sequence (or complementsthereof), respectively. PCR1 700 f can generate first amplified barcodedsecreted factor binding reagent specific oligonucleotide 742. PCR1 700 fcan comprise 1-30 cycles (e.g., 15 cycles). First amplified barcodedsecreted factor binding reagent specific oligonucleotide 742 can undergoa second round of amplification (“PCR2”) 700 g employing amplificationprimers 744 and 746 that can anneal to first universal sequence andsecond universal sequence (or complements thereof), respectively. PCR2700 g can generate second amplified barcoded secreted factor bindingreagent specific oligonucleotide 748. PCR2 700 g can add sequencingadapter 750 via an overhang in primer 746. PCR2 700 g can comprise 1-30cycles (e.g., 15 cycles). The workflow can comprise libraryamplification (“Index PCR”) 700 h. Index PCR 700 h can comprise libraryamplification of second amplified barcoded secreted factor bindingreagent specific oligonucleotide 748 with sequencing libraryamplification primers 752 and 754. Sequencing library amplificationprimers 752 and 754 can anneal to first universal sequence and seconduniversal sequence (or complements thereof) and/or sequencing adapter750. Library PCR 700 h can add sequencing adapters (e.g., P5 758 and P7764) and sample index 760 and/or 762 (e.g., i5, i7) via overhangs insequencing library amplification primers 752 and 754. Library PCRamplicons 756 can be sequenced and subjected to downstream methods ofthe disclosure. Sequencing 700 i using 150 bp×2 sequencing can revealthe cell label, the first molecular label and/or unique factoridentifier sequence (or a partial sequence of the unique factoridentifier sequence) on read 1, the unique factor identifier sequence(or a partial sequence of the unique factor identifier sequence) and/orthe second molecular label on read 2, and a sample index on index 1 readand/or index 2 read.

In some embodiments, barcoded secreted factor binding reagent specificoligonucleotide 734 can undergo a first round of amplification (“PCR1”)700 j employing amplification primers 758 and 760 that can anneal tofirst universal sequence and second universal sequence (or complementsthereof), respectively. PCR1 700 j can generate first amplified barcodedsecreted factor binding reagent specific oligonucleotide 762. PCR1 700 jcan comprise 1-30 cycles (e.g., 15 cycles). PCR1 700 j can addsequencing adapter 750 via an overhang in primer 760. The workflow cancomprise library amplification (“Index PCR”) 700 k. Index PCR 700 k cancomprise library amplification of first amplified barcoded secretedfactor binding reagent specific oligonucleotide 762 with sequencinglibrary amplification primers 764 and 766. Sequencing libraryamplification primers 764 and 766 can anneal to first universal sequenceand second universal sequence (or complements thereof) and/or sequencingadapter 750. Library PCR 700 k can add sequencing adapters (e.g., P5 758and P7 764) and sample index 760 and/or 762 (e.g., i5, i7) via overhangsin sequencing library amplification primers 764 and 766. Library PCRamplicons 768 can be sequenced and subjected to downstream methods ofthe disclosure. Sequencing 700 l using 150 bp×2 sequencing can revealthe cell label, the first molecular label and/or unique factoridentifier sequence (or a partial sequence of the unique factoridentifier sequence) on read 1, the unique factor identifier sequence(or a partial sequence of the unique factor identifier sequence) and/orthe second molecular label on read 2, and a sample index on index 1 readand/or index 2 read.

In some embodiments, the plurality of oligonucleotide barcodes areassociated with a third solid support. A second partition of theplurality of second partitions can comprise a single third solidsupport. The first partition and/or second partition can be a well or adroplet. Each oligonucleotide barcode can comprise a first universalsequence. The oligonucleotide barcode can comprise a target-bindingregion comprising a capture sequence. The target-binding region cancomprise a poly(dT) region. The secreted factor-binding reagent specificoligonucleotide can comprise a sequence complementary to the capturesequence configured to capture the secreted factor-binding reagentspecific oligonucleotide. The sequence complementary to the capturesequence can comprise a poly(dA) region. In some embodiments, theplurality of barcoded secreted factor-binding reagent specificoligonucleotides comprise a complement of the first universal sequence.The secreted factor-binding reagent specific oligonucleotide cancomprise a second universal sequence.

In some embodiments, obtaining sequence information of the plurality ofbarcoded secreted factor-binding reagent specific oligonucleotides, orproducts thereof, comprises: amplifying the plurality of barcodedsecreted factor-binding reagent specific oligonucleotides, or productsthereof, using a primer capable of hybridizing to the first universalsequence, or a complement thereof, and a primer capable of hybridizingto the second universal sequence, or a complement thereof, to generate aplurality of amplified barcoded secreted factor-binding reagent specificoligonucleotides; and obtaining sequencing data of the plurality ofamplified barcoded secreted factor-binding reagent specificoligonucleotides, or products thereof.

The secreted factor-binding reagent specific oligonucleotide cancomprise a second molecular label. In some embodiments, at least ten ofthe plurality of secreted factor-binding reagent specificoligonucleotides comprise different second molecular label sequences. Insome embodiments, the second molecular label sequences of at least twosecreted factor-binding reagent specific oligonucleotides are different,and the unique identifier sequences of the at least two secretedfactor-binding reagent specific oligonucleotides are identical. In someembodiments, the second molecular label sequences of at least twosecreted factor-binding reagent specific oligonucleotides are different,and the unique identifier sequences of the at least two secretedfactor-binding reagent specific oligonucleotides are different. In someembodiments, the number of unique first molecular label sequencesassociated with the unique factor identifier sequence for the secretedfactor-binding reagent capable of specifically binding to the at leastone secreted factor in the sequencing data indicates the number ofcopies of the at least one secreted factor secreted by each of the oneor more single cells. In some embodiments, the number of unique secondmolecular label sequences associated with the unique factor identifiersequence for the secreted factor-binding reagent capable of specificallybinding to the at least one secreted factor in the sequencing dataindicates the number of copies of the at least one secreted factorsecreted by each of the one or more single cells. In some embodiments,the method comprises determining the number of copies of the at leastone secreted factor secreted by each of the one or more single cellsbased on the number of first molecular labels and/or second molecularlabels with distinct sequences associated with the plurality of barcodedsecreted factor-binding reagent specific oligonucleotides, or productsthereof. In some embodiments, the method comprises determining thenumber of copies of the at least one secreted factor secreted by each ofthe one or more single cells based on the number of first molecularlabels and/or second molecular labels with distinct sequences associatedwith the plurality of amplified barcoded secreted factor-binding reagentspecific oligonucleotides, or products thereof. In some embodiments,obtaining the sequence information comprises attaching sequencingadaptors to the plurality of barcoded secreted factor-binding reagentspecific oligonucleotides, or products thereof. The secretedfactor-binding reagent specific oligonucleotide can be configured to bedetachable from the secreted factor-binding reagent. The method cancomprise dissociating the secreted factor-binding reagent specificoligonucleotide from the secreted factor-binding reagent.

Determining the copy number of the nucleic acid target in each of theone or more single cells can comprise determining the copy number of thenucleic acid target in each of the one or more single cells based on thenumber of first molecular labels with distinct sequences, complementsthereof, or a combination thereof, associated with the plurality ofbarcoded nucleic acid molecules, or products thereof. The method cancomprise: contacting random primers with the plurality of barcodednucleic acid molecules, each of the random primers comprises a thirduniversal sequence, or a complement thereof; and extending the randomprimers hybridized to the plurality of barcoded nucleic acid moleculesto generate a plurality of extension products. The method can compriseamplifying the plurality of extension products using primers capable ofhybridizing to the first universal sequence or complements thereof, andprimers capable of hybridizing the third universal sequence orcomplements thereof, thereby generating a first plurality of barcodedamplicons. Amplifying the plurality of extension products can compriseadding sequences of binding sites of sequencing primers and/orsequencing adaptors, complementary sequences thereof, and/or portionsthereof, to the plurality of extension products. The method can comprisedetermining the copy number of the nucleic acid target in each of theone or more single cells based on the number of first molecular labelswith distinct sequences associated with the first plurality of barcodedamplicons, or products thereof. Determining the copy number of thenucleic acid target in each of the one or more single cells can comprisedetermining the number of each of the plurality of nucleic acid targetsin each of the one or more single cells based on the number of the firstmolecular labels with distinct sequences associated with barcodedamplicons of the first plurality of barcoded amplicons comprising asequence of the each of the plurality of nucleic acid targets. Thesequence of the each of the plurality of nucleic acid targets cancomprise a subsequence of the each of the plurality of nucleic acidtargets. The sequence of the nucleic acid target in the first pluralityof barcoded amplicons can comprise a subsequence of the nucleic acidtarget.

The method can comprise amplifying the first plurality of barcodedamplicons using primers capable of hybridizing to the first universalsequence or complements thereof, and primers capable of hybridizing thethird universal sequence or complements thereof, thereby generating asecond plurality of barcoded amplicons. Amplifying the first pluralityof barcoded amplicons can comprise adding sequences of binding sites ofsequencing primers and/or sequencing adaptors, complementary sequencesthereof, and/or portions thereof, to the first plurality of barcodedamplicons. The method can comprise determining the copy number of thenucleic acid target in each of the one or more single cells based on thenumber of first molecular labels with distinct sequences associated withthe second plurality of barcoded amplicons, or products thereof. In someembodiments, the first plurality of barcoded amplicons and/or the secondplurality of barcoded amplicons comprise whole transcriptomeamplification (WTA) products.

The method can comprise synthesizing a third plurality of barcodedamplicons using the plurality of barcoded nucleic acid molecules astemplates to generate a third plurality of barcoded amplicons.Synthesizing a third plurality of barcoded amplicons can compriseperforming polymerase chain reaction (PCR) amplification of theplurality of the barcoded nucleic acid molecules. Synthesizing a thirdplurality of barcoded amplicons can comprise PCR amplification usingprimers capable of hybridizing to the first universal sequence, or acomplement thereof, and a target-specific primer. The method cancomprise obtaining sequence information of the third plurality ofbarcoded amplicons, or products thereof. Obtaining the sequenceinformation can comprise attaching sequencing adaptors to the thirdplurality of barcoded amplicons, or products thereof. The method cancomprise determining the copy number of the nucleic acid target in eachof the one or more single cells based on the number of first molecularlabels with distinct sequences associated with the third plurality ofbarcoded amplicons, or products thereof.

The nucleic acid target can comprise a nucleic acid molecule. Thenucleic acid molecule can comprise ribonucleic acid (RNA), messenger RNA(mRNA), microRNA, small interfering RNA (siRNA), RNA degradationproduct, RNA comprising a poly(A) tail, a sample indexingoligonucleotide, a cellular component-binding reagent specificoligonucleotide, or any combination thereof. In some embodiments,extending the plurality of oligonucleotide barcodes comprising extendingthe plurality of oligonucleotide barcodes using a reverse transcriptaseand/or a DNA polymerase lacking at least one of 5′ to 3′ exonucleaseactivity and 3′ to 5′ exonuclease activity. The DNA polymerase cancomprise a Klenow Fragment. The reverse transcriptase can comprise aviral reverse transcriptase (e.g., a murine leukemia virus (MLV) reversetranscriptase or a Moloney murine leukemia virus (MMLV) reversetranscriptase). In some embodiments, the first universal sequence, thesecond universal sequence, and/or the third universal sequence are thesame. In some embodiments, the first universal sequence, the seconduniversal sequence, and/or the third universal sequence are different.In some embodiments, the first universal sequence, the second universalsequence, and/or the third universal sequence comprise the binding sitesof sequencing primers and/or a sequencing adaptor, complementarysequences thereof, and/or portions thereof. In some embodiments, thesequencing adaptors comprise a P5 sequence, a P7 sequence, complementarysequences thereof, and/or portions thereof. In some embodiments, thesequencing primers comprise a Read 1 sequencing primer, a Read 2sequencing primer, complementary sequences thereof, and/or portionsthereof. In some embodiments, at least 10 of the plurality ofoligonucleotide barcodes comprise different first molecular labelsequences. In some embodiments, the plurality of oligonucleotidebarcodes each comprise a cell label. Each cell label of the plurality ofoligonucleotide barcodes can comprise at least 6 nucleotides. In someembodiments, oligonucleotide barcodes associated with the same thirdsolid support comprise the same cell label. In some embodiments,oligonucleotide barcodes associated with different third solid supportscomprise different cell labels.

Compositions and Kits

Disclosed herein include compositions (e.g., kits). The composition cancomprise: a first solid support comprising a plurality of capture probescapable of specifically binding to at least one of a plurality ofsecreted factors secreted by a single cell; and a plurality of secretedfactor-binding reagents each capable of specifically binding to asecreted factor bound by a capture probe, each of the plurality ofsecreted factor-binding reagents comprises a secreted factor-bindingreagent specific oligonucleotide comprising a unique factor identifiersequence for the secreted factor-binding reagent.

Disclosed herein include compositions (e.g., kits). The composition cancomprise: a second solid support comprising a plurality of captureprobes and a plurality of anchor probes, each of the plurality of anchorprobes is capable of specifically binding to a surface cellular target,and the capture probe is capable of specifically binding to at least oneof a plurality of secreted factors secreted by a single cell; and aplurality of secreted factor-binding reagents each capable ofspecifically binding to a secreted factor bound by a capture probe, eachof the plurality of secreted factor-binding reagents comprises asecreted factor-binding reagent specific oligonucleotide comprising aunique factor identifier sequence for the secreted factor-bindingreagent.

The secreted factor-binding reagent specific oligonucleotide cancomprise a second molecular label sequence. The second molecular labelsequence can be 2-20 nucleotides in length. In some embodiments, thesecond molecular label sequences of at least two secreted factor-bindingreagent specific oligonucleotides are different, and wherein the uniqueidentifier sequences of the at least two secreted factor-binding reagentspecific oligonucleotides are identical. In some embodiments, the secondmolecular label sequences of at least two secreted factor-bindingreagent specific oligonucleotides are different, and wherein the uniqueidentifier sequences of the at least two secreted factor-binding reagentspecific oligonucleotides are different.

The secreted factor-binding reagent specific oligonucleotide cancomprise a second universal sequence. The second universal sequence cancomprise a binding site of a sequencing primers and/or sequencingadaptor, complementary sequences thereof, and/or portions thereof. Thesequencing adaptor can comprise a P5 sequence, a P7 sequence,complementary sequences thereof, and/or portions thereof. The sequencingprimer can comprise a Read 1 sequencing primer, a Read 2 sequencingprimer, complementary sequences thereof, and/or portions thereof.

The cellular component-binding reagent specific oligonucleotide cancomprise a poly(dA) region. The secreted factor-binding reagent specificoligonucleotide can comprise an alignment sequence adjacent to thepoly(dA) region. The alignment sequence can be one or more nucleotidesin length. The alignment sequence can be two or more nucleotides inlength. The alignment sequence can comprise a guanine, a cytosine, athymine, a uracil, or a combination thereof. The alignment sequence cancomprise a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence,a poly(dU) sequence, or a combination thereof. The alignment sequencecan be 5′ to the poly(dA) region.

The secreted factor-binding reagent specific oligonucleotide can beassociated with the secreted factor-binding reagent through a linker.The linker can comprise a carbon chain. The carbon chain can comprise2-30 carbons. The carbon chain can comprise 12 carbons. The linker cancomprise 5′ amino modifier C12 (5AmMC12), or a derivative thereof. Thesecreted factor-binding reagent specific oligonucleotide can be attachedto the secreted factor-binding reagent. The secreted factor-bindingreagent specific oligonucleotide can be covalently attached to thesecreted factor-binding reagent. The secreted factor-binding reagentspecific oligonucleotide can be non-covalently attached to the secretedfactor-binding reagent. The secreted factor-binding reagent specificoligonucleotide can be conjugated to the secreted factor-bindingreagent. The secreted factor-binding reagent specific oligonucleotidecan be conjugated to the secreted factor-binding reagent through achemical group selected from the group consisting of a UV photocleavablegroup, a streptavidin, a biotin, an amine, and a combination thereof.

The composition can comprise a DNA polymerase (e.g., a Klenow Fragment)lacking at least one of 5′ to 3′ exonuclease activity and 3′ to 5′exonuclease activity. The composition can comprise a reversetranscriptase, such as a viral reverse transcriptase (e.g., murineleukemia virus (MLV) reverse transcriptase or a Moloney murine leukemiavirus (MMLV) reverse transcriptase). The composition can comprise abuffer, a cartridge, or both. The composition can comprise a pluralityof oligonucleotide barcodes. The plurality of oligonucleotide barcodesare associated with a third solid support. The composition can comprisethird solid supports.

Terminology

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

One skilled in the art will appreciate that, for this and otherprocesses and methods disclosed herein, the functions performed in theprocesses and methods can be implemented in differing order.Furthermore, the outlined steps and operations are only provided asexamples, and some of the steps and operations can be optional, combinedinto fewer steps and operations, or expanded into additional steps andoperations without detracting from the essence of the disclosedembodiments.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise. Any reference to “or” herein isintended to encompass “and/or” unless otherwise stated.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims.

What is claimed is:
 1. A method for measuring the number of copies of asecreted factor secreted by a single cell, comprising: contacting one ormore single cells with a first plurality of first solid supports,wherein the one or more single cells are capable of secreting aplurality of secreted factors, wherein each first solid supportcomprises a plurality of capture probes capable of specifically bindingto at least one of the plurality of secreted factors secreted by asingle cell; contacting the first solid support with a plurality ofsecreted factor-binding reagents each capable of specifically binding toa secreted factor bound by a capture probe, wherein each of theplurality of secreted factor-binding reagents comprises a secretedfactor-binding reagent specific oligonucleotide comprising a uniquefactor identifier sequence for the secreted factor-binding reagent;contacting a plurality of oligonucleotide barcodes with the secretedfactor-binding reagent specific oligonucleotides for hybridization,wherein the oligonucleotide barcodes each comprise a first molecularlabel; extending the plurality of oligonucleotide barcodes hybridized tothe secreted factor-binding reagent specific oligonucleotides togenerate a plurality of barcoded secreted factor-binding reagentspecific oligonucleotides each comprising a sequence complementary to atleast a portion of the unique factor identifier sequence and the firstmolecular label; and obtaining sequence information of the plurality ofbarcoded secreted factor-binding reagent specific oligonucleotides, orproducts thereof, to determine the number of copies of the at least onesecreted factor secreted by each of the one or more single cells.
 2. Themethod of claim 1, wherein contacting one or more single cells with afirst plurality of first solid supports comprises partitioning the oneor more single cells and the first plurality of first solid supports toa plurality of first partitions, wherein a first partition of theplurality of first partitions comprises a single cell of the one or moresingle cells and a single first solid support of the first plurality offirst solid supports.
 3. The method of claim 2, comprising, prior tocontacting the first solid support with a plurality of secretedfactor-binding reagents: pooling the single first solid supports fromeach first partition of the plurality of first partitions to generate asecond plurality of first solid supports.
 4. The method of claim 3,wherein contacting the first solid support with a plurality of secretedfactor-binding reagents comprises contacting the second plurality offirst solid supports with the plurality of secreted factor-bindingreagents.
 5. The method of claim 4, comprising, after contacting thesecond plurality of first solid supports with the plurality of secretedfactor-binding reagents, removing one or more secreted factor-bindingreagents of the plurality of secreted factor-binding reagents that arenot contacted with the second plurality of first solid supports togenerate a third plurality of first solid supports, wherein removing theone or more secreted factor-binding reagents not contacted with thesecond plurality of first solid supports comprises: removing the one ormore secreted factor-binding reagents not contacted with the respectiveat least one of the secreted factor bound by a capture probe.
 6. Themethod of claim 1, wherein contacting a plurality of oligonucleotidebarcodes with the secreted factor-binding reagent specificoligonucleotides for hybridization comprises: partitioning the thirdplurality of first solid supports to a plurality of second partitions,wherein a second partition of the plurality of second partitionscomprises a single first solid support from the third plurality of firstsolid supports; and in the second partition comprising the single firstsolid support, contacting a plurality of oligonucleotide barcodes withthe secreted factor-binding reagent specific oligonucleotides forhybridization.
 7. A method for measuring the number of copies of asecreted factor secreted by a single cell and the number of copies of anucleic acid target in a single cell, comprising: contacting one or moresingle cells with a first plurality of second solid supports to form oneor more single cells associated with a second solid support, wherein theone or more single cells comprise a surface cellular target and copiesof a nucleic acid target, wherein the one or more single cells arecapable of secreting a plurality of secreted factors, wherein eachsecond solid support comprises a plurality of capture probes and aplurality of anchor probes, wherein each of the plurality of anchorprobes is capable of specifically binding to the surface cellulartarget, and wherein the capture probe is capable of specifically bindingto at least one of the plurality of secreted factors secreted by asingle cell; contacting the one or more single cells associated with asecond solid support with a plurality of secreted factor-bindingreagents capable of specifically binding to a secreted factor bound by acapture probe, wherein each of the plurality of secreted factor-bindingreagents comprises a secreted factor-binding reagent specificoligonucleotide comprising a unique factor identifier sequence for thesecreted factor-binding reagent; contacting a plurality ofoligonucleotide barcodes with the secreted factor-binding reagentspecific oligonucleotides and the copies of the nucleic acid target forhybridization, wherein the oligonucleotide barcodes each comprise afirst molecular label; extending the plurality of oligonucleotidebarcodes hybridized to the copies of a nucleic acid target to generate aplurality of barcoded nucleic acid molecules each comprising a sequencecomplementary to at least a portion of the nucleic acid target and thefirst molecular label; extending the plurality of oligonucleotidebarcodes hybridized to the secreted factor-binding reagent specificoligonucleotides to generate a plurality of barcoded secretedfactor-binding reagent specific oligonucleotides each comprising asequence complementary to at least a portion of the unique factoridentifier sequence and the first molecular label; obtaining sequenceinformation of the plurality of barcoded nucleic acid molecules, orproducts thereof, to determine the copy number of the nucleic acidtarget in each of the one or more single cells; and obtaining sequenceinformation of the plurality of barcoded secreted factor-binding reagentspecific oligonucleotides, or products thereof, to determine the numberof copies of the at least one secreted factor secreted by each of theone or more single cells.
 8. The method of claim 1, wherein the one ormore single cells comprises T cells, B cells, tumor cells, myeloidcells, blood cells, normal cells, fetal cells, maternal cells, or amixture thereof.
 9. The method of claim 1, wherein the at least onesecreted factor comprises: (i) a lymphokine, an interleukin, achemokine, or any combination thereof; (ii) a cytokine, a hormone, amolecular toxin, or any combination thereof and/or (iii) a nerve growthfactor, a hepatic growth factor, a fibroblast growth factor, a vascularendothelial growth factor, a platelet-derived growth factor, atransforming growth factor, an osteoinductive factor, an interferon, acolony stimulating factor, or any combination thereof.
 10. The method ofclaim 1, wherein the secreted factor-binding reagent and the captureprobe are capable of binding to distinct epitopes of the same secretedfactor.
 11. The method of claim 1, wherein one or more of the secretedfactor-binding reagents, the capture probe, and the anchor probecomprise an antibody or fragment thereof, wherein the antibody orfragment thereof comprises a Fab, a Fab′, a F(ab′)₂, a Fv, a scFv, adsFv, a diabody, a triabody, a tetrabody, a monoclonal antibody, amultispecific antibody formed from antibody fragments, a single-domainantibody (sdAb), a single chain comprising complementary scFvs (tandemscFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linkedFv, a dual variable domain immunoglobulin (DVD-Ig) binding protein or ananobody, an aptamer, an affibody, an affilin, an affitin, an affimer,an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitzdomain peptide, a monobody, or any combination thereof.
 12. The methodof claim 1, wherein the surface cellular target comprises acarbohydrate, a lipid, a protein, an extracellular protein, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an intracellular protein, or any combination thereof.
 13. Themethod of claim 6, wherein the plurality of oligonucleotide barcodes areassociated with a third solid support, wherein the first partitionand/or second partition is a well or a droplet, and wherein a secondpartition of the plurality of second partitions comprises a single thirdsolid support.
 14. The method of claim 1, wherein each oligonucleotidebarcode comprises a first universal sequence, and wherein the pluralityof barcoded secreted factor-binding reagent specific oligonucleotidescomprise a complement of the first universal sequence.
 15. The method ofclaim 1, wherein the secreted factor-binding reagent specificoligonucleotide comprises a second universal sequence, and whereinobtaining sequence information of the plurality of barcoded secretedfactor-binding reagent specific oligonucleotides, or products thereof,comprises: amplifying the plurality of barcoded secreted factor-bindingreagent specific oligonucleotides, or products thereof, using a primercapable of hybridizing to the first universal sequence, or a complementthereof, and a primer capable of hybridizing to the second universalsequence, or a complement thereof, to generate a plurality of amplifiedbarcoded secreted factor-binding reagent specific oligonucleotides; andobtaining sequencing data of the plurality of amplified barcodedsecreted factor-binding reagent specific oligonucleotides, or productsthereof.
 16. The method of claim 1, wherein the secreted factor-bindingreagent specific oligonucleotide comprises a second molecular label,wherein at least ten of the plurality of secreted factor-binding reagentspecific oligonucleotides comprise different second molecular labelsequences, and wherein: (i) the second molecular label sequences of atleast two secreted factor-binding reagent specific oligonucleotides aredifferent, and wherein the unique identifier sequences of the at leasttwo secreted factor-binding reagent specific oligonucleotides areidentical; or (ii) the second molecular label sequences of at least twosecreted factor-binding reagent specific oligonucleotides are different,and wherein the unique identifier sequences of the at least two secretedfactor-binding reagent specific oligonucleotides are different.
 17. Themethod of claim 1, wherein the number of unique first molecular labelsequences associated with the unique factor identifier sequence for thesecreted factor-binding reagent capable of specifically binding to theat least one secreted factor in the sequencing data indicates the numberof copies of the at least one secreted factor secreted by each of theone or more single cells.
 18. The method of claim 16, wherein the numberof unique second molecular label sequences associated with the uniquefactor identifier sequence for the secreted factor-binding reagentcapable of specifically binding to the at least one secreted factor inthe sequencing data indicates the number of copies of the at least onesecreted factor secreted by each of the one or more single cells. 19.The method of claim 1, comprising determining the number of copies ofthe at least one secreted factor secreted by each of the one or moresingle cells based on the number of first molecular labels and/or secondmolecular labels with distinct sequences associated with the pluralityof barcoded secreted factor-binding reagent specific oligonucleotides,or products thereof.
 20. The method of claim 16, comprising determiningthe number of copies of the at least one secreted factor secreted byeach of the one or more single cells based on the number of firstmolecular labels and/or second molecular labels with distinct sequencesassociated with the plurality of amplified barcoded secretedfactor-binding reagent specific oligonucleotides, or products thereof.21. The method of claim 1, wherein the first solid support and/or thesecond solid support has the dimensions of a cell, wherein the cell is amammalian cell, a yeast cell, an insect cell, a plant cell, a bacterialcell, or any combination thereof.
 22. A composition comprising: a firstsolid support comprising a plurality of capture probes capable ofspecifically binding to at least one of a plurality of secreted factorssecreted by a single cell; and a plurality of secreted factor-bindingreagents each capable of specifically binding to a secreted factor boundby a capture probe, wherein each of the plurality of secretedfactor-binding reagents comprises a secreted factor-binding reagentspecific oligonucleotide comprising a unique factor identifier sequencefor the secreted factor-binding reagent.
 23. The composition of claim22, wherein the secreted factor comprises: (i) a lymphokine, aninterleukin, a chemokine, or any combination thereof; (ii) a cytokine, ahormone, a molecular toxin, or any combination thereof and/or (iii) anerve growth factor, a hepatic growth factor, a fibroblast growthfactor, a vascular endothelial growth factor, a platelet-derived growthfactor, a transforming growth factor, an osteoinductive factor, aninterferon, a colony stimulating factor, or any combination thereof. 24.The composition of claim 22, wherein the first solid support has thedimensions of a cell, wherein the cell is a mammalian cell, a yeastcell, an insect cell, a plant cell, a bacterial cell, or any combinationthereof.